Treatment of male androgen deficiency symptoms or diseases with sex steroid precursor combined with serm

ABSTRACT

Novel methods for prevention, reduction or elimination of the incidence of male androgen deficiency symptoms or diseases including male hypogonadism-associated symptoms and diseases associated with low serum testosterone and/or low DHEA or low total androgens in susceptible warm-blooded animals including humans involving administration of an amount of a sex steroid precursor, particularly dehydroepiandrosterone (DHEA) and a selective estrogen receptor modulator (SERM) (particularly acolbifene), an antiestrogen or a prodrug of the two. The symptoms or diseases are loss of libido, erectile dysfunction, tiredness, loss of energy, depression, bone loss, muscle loss, muscle weakness, fat accumulation, memory loss, cognition loss, Alzheimer&#39;s disease, dementia, loss of body hair, fertility problems, insomnia, gynecomastia, anemia, hot flushes, sweats, decreased sense of well-being, obesity, osteoporosis, hypercholesterolemia, hyperlipidemia, atherosclerosis, hypertension, insulin resistance, cardiovascular disease and type 2 diabetes. Pharmaceutical compositions for delivery of active ingredient(s) and kit(s) useful to the invention are also disclosed.

FIELD OF THE INVENTION

The present invention relates to a novel treatment of low totalandrogens accompanied by one or more symptoms classically attributed tomale hypogonadism or low testosterone. The number of individuals over 65years of age has increased more than 10-fold compared with the 1990s(Shigehara and Namiki 2011). In the aging process, low testosterone isoften accompanied by decreased sense of well-being, depression,decreased libido and increased erectile dysfunction (Lunenfeld andNieschlag 2007). The decrease in serum testosterone levels associatedwith aging has been called late-onset hypogonadism (LOH) (Wang,Nieschlag et al. 2009b). The diagnosis of male hypogonadism usuallycombined symptomatology in addition to low serum testosterone reportedas below 2.0-3.5 ng/mL.

The precise threshold testosterone level below which symptoms ofandrogen deficiency and adverse health outcomes occur is not known andmay be age-dependent (Kelleher, Conway et al. 2004; Zitzmann, Faber etal. 2006; Hall, Esche et al. 2008).

At a threshold of 3.0 ng testosterone/mL, symptoms occur more below thisvalue (Kelleher, Conway et al. 2004; Zitzmann, Faber et al. 2006;Bhasin, Cunningham et al. 2010). The guidelines from the US EndocrineSociety have defined LOH as a serum testosterone less than 2.0 ng/mL inconjunction with one or more signs and symptoms of classicalhypogonadism (Bhasin, Cunningham et al. 2006). The American Society ofAndrology recommends less than 3.0 ng/mL in symptomatic men (AmericanSociety of Andrology 2006). On the other hand, according to theInternational Society for the Study of the Aging Male (ISSAM),symptomatic aged men should be considered hypogonadal at less than 3.50ng testosterone/mL (Wang, Nieschlaq et al. 2009a).

In parallel, the testosterone concentration below which testosteroneadministration improves outcomes is unclear and may vary amongindividuals and among target organs. Therefore, the available evidencedoes not support use of an arbitrary threshold for testosterone levelbelow which clinical androgen deficiency occurs and that confirms thediagnosis of hypogonadism in all patients (Bhasin, Cunningham et al.2006).

A correlation between low physical vigor and low serum testosterone hasbeen repeatedly low (Xu, Gouras et al. 1998; Travison, Morley et al.2006). It is also quite possible, as mentioned above, that variousthresholds exist for the various androgen-dependent targets (Bhasin,Woodhouse et al. 2005; Gray, Singh et al. 2005; Zitzmann, Faber et al.2006; Shigehara and Namiki 2011).

A novel component at the basis of the present invention is thatconsideration should also be given to isolated or combined lowintracrine peripheral formation of androgens from low serumdehydroepiandrosterone (DHEA) with a symptomatology similar to thatattributed to hypogonadism. Accordingly, the DHEA-derived androgenmetabolites, especially androsterone glucuronide (ADT-G), can bemeasured as described (Labrie, Bélanger et al. 2006). The normal valuesof dehydroepiandrosterone (DHEA) and androgen metabolite glucuronides,namely ADT-G (estimate of total androgenicity) and other androgens andmetabolites can be seen in (Labrie, Cusan et al. 2009; Labrie 2010b;Ohlsson, Labrie et al. 2010; Labrie 2011; O'Connor, Lee et al. 2011).Values of serum DHEA below 2.0 ng/mL by themselves can be considered lowwith normal testosterone but the concentration of serum testosteronemust also be taken into consideration and the symptoms of low totalandrogens results from the combination of low testosterone and/or lowDHEA resulting in low total androgens reflected by low androgenmetabolites. Serum ADT-G below 25 ng/mL can be considered a parameter oflow total hypoandrogenecity (Labrie, Diamond et al. 1997b).

Male hypogonadism can represent deficiency in spermatogenesis or adeficiency in testicular testosterone secretion. This second part willbe involved in the present invention (please see (Corona, Rastrelli etal. 2012) for more details).

Typically, late-onset hypogonadism (LOH) appearing in the aging malecombines low serum testosterone with one or more symptoms ofhypoandrogenecity. However, since up to 50% of total androgens derivefrom DHEA, low DHEA can be as responsible as low testosterone of thesigns and symptoms of hypogonadism.

Consequently, the signs and symptoms of hypogonadism and/or lowperipheral androgen formation can be appropriate conditions for therapy.Free testosterone can also be measured according to Vermeulen's formulawww.issam.ch/freetesto.htm, but is not usually very informative.

In addition to testosterone, the testis, through the action of aromatasesecretes the estrogens estrone and estradiol (FIG. 1). The secretion ofluteinizing hormone (LH) by the anterior pituitary gland is stimulatedby the pulsatile secretion of GnRH (Gonadotropin-Releasing Hormone) fromthe hypothalamus while both testosterone and estradiol exert globalinhibitory effects at the hypothalamo-pituitary level on LH secretion(Corona, Rastrelli et al. 2012). LH then stimulates testosteronesecretion by the Leydig cells in the testis (FIG. 1).

The guidelines of the US Endocrine Society recommend testosteronetreatment only in men with “consistent symptoms and signs andunequivocally low serum testosterone levels”. However, it has been foundthat only half the men receiving testosterone replacement therapy werediagnosed with male hypogonadism. In fact, 34% were treated for fatigue,31% for erectile dysfunction and 12% for psychosexual dysfunction(Baillarqeon, Urban et al. 2013).

As mentioned above, it must be considered that up to 50% of totalandrogens in men are made locally in peripheral tissues from DHEA thatdecreases with age by as much as 80% on average in men aged 75 years ormore (Labrie, Bélanger et al. 1997b), thus providing a reason why lowDHEA has at least an equal role compared to low serum testosterone toexplain the symptoms and signs so-far attributed to male hypogonadism(Labrie, Bélanger et al. 1997a).

The Endocrine Society has a Clinical Practice Guideline on testosteronetherapy, namely Testosterone Therapy in Men with Androgen DeficiencySyndrome (2006; revised 2010) at www.endocrine.orq. It includes therevised recommendations on the Prostate Specific Antigen exclusioncriteria and PSA follow-up guidance.

Low testosterone can be accompanied by any single or a combination ofthe following signs or symptoms:

-   -   loss of libido (interest in sex)    -   difficulty in getting an erection (erectile dysfunction)    -   tiredness and lack of energy (loss of energy, energy loss)    -   depression    -   loss of bone (decreased bone mineral density and increased risk        of fracture)    -   loss of muscle and muscle weakness    -   loss of body hair    -   fertility problems

Additional benefits such as treatment or reduction of the likelihood orrisk of acquiring the following medical problems, namelyhypercholesterolemia, hyperlipidemia, atherosclerosis, hypertension,Alzheimer's disease, loss of memory, loss of cognition, dementia,insomnia, cardiovascular diseases, insulin resistance, Type 2 diabetesand obesity (especially abdominal obesity) (Comhaire 2000; Ding, Song etal. 2006; Khaw, Dowsett et al. 2007; Bassil, Alkaade et al. 2009;Zitzmann 2009) are also provided by treatment with the invention.

Low serum testosterone in men is associated with low muscle mass,decreased muscle strength and poor mobility (Roy, Blackman et al. 2002;Schaap, Pluijm et al. 2005). Testosterone supplementation in healthyolder men increases muscle mass and strength and leg power, these beingimportant factors of mobility (Bhasin, Storer et al. 1996; Sih, Morleyet al. 1997; Snyder, Peachey et al. 1999; Storer, Magliano et al. 2003;Bhasin, Woodhouse et al. 2005; Page, Amory et al. 2005).

Symptoms/signs of androgen deficiency in aging males can be as stated inthe Clinical Practice Guideline of the Endocrine Society (Bhasin,Cunningham et al. 2006).

TABLE 1A Symptoms and signs suggestive of androgen deficiency in agingmen   Reduced sexual desire (libido) and activity Decreased spontaneouserections Breast discomfort, gynecomastia Loss of body (axillary andpubic) hair, reduced shaving Very small or shrinking testes (especially<5 mL) Inability to father children, low or zero sperm counts Heightloss, low trauma fracture, low bone mineral density Reduced muscle bulkand strength Hot flushes, sweats

TABLE 1B Other symptoms and signs associated with androgen deficiencythat are less specific than those in Table 1A   Decreased energy,motivation, initiative, aggressiveness, self-confidence Feeling sad orblue, depressed mood, dysthymia Poor concentration and memory Sleepdisturbance, increased sleepiness Mild anemia (normochromic, normocytic,in the female range) Increased body fat, body mass index Diminishedphysical or work performance (Bhasin, Cunningham et al. 2006)

Aging itself is often associated with a decline in sexual functioning inmen (Vermeulen 2003; Ebert, Jockenhovel et al. 2005).

The diagnosis of male hypogonadism can be helped by the ANDROTEST(Corona, Jannini et al. 2006; Corona, Mannucci et al. 2006).Differential diagnosis can also be helped by the information provided by(Corona, Rastrelli et al. 2012). LOH has also been defined by thepresence of at least three sexual symptoms associated with a totaltestosterone level of less than 3.2 ng/mL (Wu, Tajar et al. 2010). Inthat study performed in a random population sample of 3369 men aged 40to 79 years, differences between asymptomatic and symptomatic men inrelation with serum testosterone were minimal. One possible explanationcould be, as indicated above, that serum testosterone is not theexclusive source of androgenic activity which is, as mentioned above, upto 50% from DHEA-derived androgens (Labrie, Dupont et al. 1985; Labrie2011).

The physiological role of testosterone in male sexual behavior is poorlyunderstood. Many studies with attempts to correlate male sexual behaviorand the concentration of serum testosterone have given conflictingresults. There are wide variations between serum testosterone levels anderectile dysfunction (Salmimies, Kockott et al. 1982; Gooren 1987;Bhasin, Cunningham et al. 2006; Traish, Guay et al. 2009). It remains,however, that low serum testosterone has become standard clinicalpractice in the evaluation of sexual disorders in men.

Diagnosis of late-onset male hypogonadism can be helped byquestionnaires, although clinical evaluation of the total clinicalpicture is of major importance. The instruments which can be used are,without limitation, Androgen Deficiency in Aging Males (ADAM) (Morley,Charlton et al. 2000), the Aging Males Symptoms (AMS) Rating Scale(Moore, Huebler et al. 2004) and the Massachusetts Male Ageing Study(MMAS) Questionnaire (Smith, Feldman et al. 2000). Diagnosis can behelped with the Brief Sexual Function Inventory (BSFI) (O'Leary, Fowleret al. 1995). The instrument covers sexual drive (two items), erection(three items), ejaculation (two items), perception of problems in eacharea (three items) and overall satisfaction (one item).

There is an emerging medication for the treatment of male hypogonadism(see the two following recent reviews: (Corona, Rastrelli et al. 2012;Kim, Crosnoe et al. 2013)). In addition to the existing exogenoustestosterone treatment, clinical data with selective estrogen receptormodulators (SERMs) are available. A SERM binds to the estrogen receptorin the hypothalamus and pituitary gland in competition with estradiol.The neutralization of inhibitory action of estradiol in the hypothalamusincreases GnRH (gonadotropin-releasing hormone) secretion whichstimulates LH secretion which increases testosterone production by thetestes. Several studies with clomiphene citrate have been performed.Clomiphene citrate increases serum testosterone levels in the blood likethe use of testosterone gels (Taylor and Levine 2010). Clomiphenecitrate improves sexual function in hypogonadal men (Guav, Jacobson etal. 2003). Clomiphene citrate improves the testosterone-estradiol ratioin hypogonadal men (Shabsigh, Kang et al. 2005). Clomiphene citrateincreases circulating testosterone and improves severalhypogonadism-related symptoms (decreased libido, lack of energy) inyoung hypogonadal men (Katz, Nabulsi et al. 2012).

Enclomiphene (Androxal; Repros) is under development for malehypogonadism and infertility. Patents literature also indicates thatSERMs or antiestrogens could be useful for male androgen deficiencyincluding male hypogonadism (US 2006/0293294, US 2009/0215733, WO01/91744, WO 03/072092, WO 2006/024689 and WO 2013/123218) and incombination with other active agents (US 2007/0078091 and WO2013/130832). Other classes of compounds have been suggested to treatmale hypogonadism, namely gonadotropins, 5α-reductase inhibitors,testosterone precursors, non-aromatizable androgens, aromataseinhibitors, selective estrogen receptor β agonists and selectiveandrogen receptor modulators (SARMs). Gonadotropin therapy remains oneof the few effective treatments for infertility in men with secondaryhypogonadism (Liu, Baker et al. 2009; Farhat, Al-zidjali et al. 2010).Human chorionic gonadotropin is an LH analogue that stimulates Leydigcell production of testosterone and it can be derived from urine as wellas recombinant sources.

In particular, the treatment includes the administration of a precursorof sex steroids in combination with a cell-specific selective estrogenreceptor modulator (SERM), in particular acolbifene.

The invention also provides kits and pharmaceutical compositions forpracticing the foregoing combination.

It is known that a large number of diseases, conditions and undesirablesymptoms respond favorably to administering exogenous sex steroids, orprecursors thereof. For example, estrogens are believed to decrease therate of bone loss while androgens have been shown to build bone mass bystimulating bone formation.

Long-term testosterone treatment in hypogonadal men improves metabolicsyndrome components. It reduced total cholesterol, low-densitylipoprotein cholesterol, tryglycerides and increased HDL cholesterollevels. It also reduced blood glucose levels (Traish, Haider et al.2013).

Treatment with dihydrotestosterone (DHT) for 2 years had no effect onprostate volume but decreased fat mass, increased lean mass, suppressedserum testosterone and decreased spinal bone mineral density, probablydue to inhibition of LH secretion. Many other studies have shown thebenefits of androgen replacement therapy with no significant change ofprostatic volume or urinary symptoms (Sih, Morley et al. 1997; Kenny,Prestwood et al. 2001; Marks, Mazer et al. 2006; Saad, Gooren et al.2008; Takao, Tsujimura et al. 2009). In a 10-year study with oraltestosterone undecanoate, no increase in prostate size and no evidenceof cancer was noted (Gooren 1994).

In hypogonadal men, even an improvement of lower urinary tract symptomswas observed (Pechersky, Mazurov et al. 2002), for review see (Amano,Imao et al. 2010; Shigehara and Namiki 2011). Oral testosteroneundecanoate replacement for 8 months at doses of 40 to 160 mg/day didnot change the prostate size nor showed deterioration of voidingsymptoms (Franchi F, Luisi M et al. 1978). A study where 100 mgtestosterone enanthate was injected weekly for 3 months similarly didnot change prostate volume or post voiding residual volume (Tenover1992).

In another study, androgen replacement therapy for 8 months increasedprostate volume by 18% with no change in uroflowmetry data (Holmang,Marin et al. 1993). No difference in prostate volume was observed inanother study (Behre, Bohmeyer et al. 1994).

Reduced libido and erectile dysfunction are considered as being the mostproeminent symptoms of hypogonadism in men (Harman, Metter et al. 2001;Matsumoto 2002). In the Massachusetts Male Aging Study, the prevalenceof complete erectile dysfunction increased 3-fold from 5% to 15% betweenthe ages 40 and 70 years (Morley 2003).

In the European Male Aging Study (EMAS), on the other hand, acorrelation was found between low serum testosterone and the symptomspoor morning erection, low sexual desire and erectile dysfunction(testosterone range 2.3 to 3.7 ng/mL) leading to the LOH (Late-OnsetHypogonadism) definition in men having the 3 symptoms and serumtestosterone less than 3.2 ng/mL or 11 nmole per liter (Wu, Taiar et al.2010). Testosterone controls gonadotropin secretion, masculinizationduring sexual maturation, induction and maintenance of sperm production,as well as libido and sexual function.

Both estrogens derived from androgens and androgens themselves exert aglobal negative effect on GnRH/LH secretion (FIG. 1). Estradiol, whilebeing at much lower concentrations in the blood, is an efficientinhibitor of GnRH/LH secretion.

Serum testosterone levels vary significantly as a result of circadianand circannual rhythms, episodic secretion, and measurement variations.Testosterone concentrations may be affected by illness and certainmedications (e.g. opiates and glucocorticoids).

In the TOM trial performed in men older than 65 years with chronicconditions and limitations in mobility, twice as many adverse events(AEs) were reported in the testosterone gel versus the placebo groups(Basaria, Coviello et al. 2010). In that relatively small group(testosterone in older men with mobility limitations, TOM) of 209 menwith serum testosterone of 1.0 to 3.5 ng/mL with a high prevalence ofchronic disease, namely hypertension, hyperlipidemia, diabetes andobesity, a higher incidence of cardiovascular events in the testosteronegel group stopped the trial. Greater improvement of leg-press andchest-press strength and in stair climbing while carrying a load wasseen in the testosterone-treated versus placebo groups (Basaria,Coviello et al. 2010). The risk of cardiovascular AEs was greater intestosterone-treated men.

Testosterone replacement therapy is also associated with infertility asside effect due to decreased sperm count as well as decrease intesticular-size.

Testosterone injections have the advantage of low cost but have thedisadvantage of non physiological peak and trough levels over theweekly, bi-weekly or long term dosing regimen.

In a group of 8709 Veteran Administration patients with serumtestosterone <3.0 ng/mL, after a median of 531 days post coronography,1223 of them started testosterone therapy (Vigen, O'Donnell et al.2013). In that retrospective observational study, the rates of deaths at3 years were 15.4% vs 18.5% in the control and testosterone groups,respectively. As stated, “this signal warrants cautions testosteroneprescribing . . . ” (Cappola 2013).

Metaanalysis of testosterone therapy trials, except the TOM trial,however, did not demonstrate adverse cardiovascular events (Calof, Singhet al. 2005; Haddad, Kennedy et al. 2007; Fernandez-Balsells, Murad etal. 2010).

Testosterone replacement therapy has been associated with increasedsexual functioning and mood (Seftel, Mack et al. 2004; Wanq, Cunninghamet al. 2004).

In addition to improving sexual function (Wanq, Swerdloff et al. 2000;Isidori, Giannetta et al. 2005; Bolona, Uraga et al. 2007), theadministration of testosterone to men with symptomatic androgendeficiency increases bone mineral density (Snyder, Peachey et al. 2000;Isidori, Giannetta et al. 2005) increases fat-free mass (Isidori, Caprioet al. 1999; Snyder, Peachey et al. 2000; Isidori, Giannetta et al.2005) and strength (Sih, Morley et al. 1997), improves insulinresistance (Jones and Saad 2009; Jones, Arver et al. 2011) and improvesthe lipid profile (Marin, Holmang et al. 1993; Jones and Saad 2009;Jones, Arver et al. 2011).

A significant problem with testosterone replacement therapy is that itsuppresses testicular endogenous testosterone secretion and can resultin azoospermia or impairment of spermatogenesis as indicated by thelabeling accepted by the Food and Drug Administration (Kim, Crosnoe etal. 2013). Exogeneous testosterone inhibits thehypothalamo-pituitary-testicular axis and can result in infertility.Intramuscular testosterone has even been studied as a contraceptiveagent (Liu, Swerdloff et al. 2006). In the present invention, the lowtesticular testosterone formation secondary to inhibition of LHsecretion is avoided by the use of a SERM, in particular acolbifene thatstimulates LH secretion instead of blocking endogenous LH and,secondarily, testosterone secretion.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method ofpreventing, reducing or eliminating the incidence of male androgendeficiency symptoms or diseases including male hypogonadism-associatedsymptoms and diseases due to low testosterone and/or low peripheralandrogen formation.

It is another object to provide methods of preventing, reducing oreliminating the incidence of loss of libido, erectile dysfunction,tiredness, loss of energy, depression, bone loss, muscle loss, muscleweakness, fat accumulation, memory loss, cognition loss, Alzheimer'sdisease, dementia, loss of body hair, fertility problems, insomnia,gynecomastia, anemia, hot flushes, sweats, decreased sense ofwell-being, obesity, osteoporosis, hypercholesterolemia, hyperlipidemia,atherosclerosis, hypertension, insulin resistance, cardiovasculardisease and type 2 diabetes.

It is another object to provide methods of reducing the risk of the malepatients acquiring breast cancer.

It is another object to provide kits and pharmaceutical compositionssuitable for use in the above methods. Preferably, these products arepackaged with directions for using the contents thereof for preventing,reducing or eliminating the incidence of male androgen deficiencysymptoms or diseases including male hypogonadism-associated symptoms anddiseases.

It is another object to provide kits and pharmaceutical compositionssuitable for use in the above methods. Preferably, these products arepackaged with directions for using the contents thereof for preventing,reducing or eliminating the incidence of loss of libido, erectiledysfunction, tiredness, loss of energy, depression, bone loss, muscleloss, muscle weakness, fat accumulation, memory loss, cognition loss,Alzheimer's disease, dementia, loss of body hair, fertility problems,insomnia, gynecomastia, anemia, hot flushes, sweats, decreased sense ofwell-being, obesity, osteoporosis, hypercholesterolemia, hyperlipidemia,atherosclerosis, hypertension, insulin resistance, cardiovasculardisease and type 2 diabetes.

In one embodiment, the invention provides a method of preventing,reducing or eliminating the incidence of male androgen deficiencysymptoms or diseases including male hypogonadism-associated symptoms anddiseases, said method comprising administering to male patient in needof said prevention, reduction or elimination, a therapeuticallyeffective amount of a sex steroid precursor or prodrug thereof inassociation with a therapeutically effective amount of a selectiveestrogen receptor modulator or an antiestrogen or prodrug of either.

It is preferred that the sex steroid precursor is selected from thegroup consisting of dehydroepiandrosterone,dehydroepiandrosterone-sulfate, androst-5-ene-3β,17β-diol,4-androstene-3,17-dione, and a prodrug of any of the foregoingadditional agents.

It is preferred that the selective estrogen receptor modulator isselected from the group comprising of Tamoxifen, Toremifene, CC 8490,SERM 3471, HMR 3339, HMR 3656, Raloxifene, LY 335124, LY 326315,Arzoxifene (LY 353381), Pipendoxifene (ERA 923), Bazedoxifene (TSE 424,WAY 140424), Oporia (Lasofoxifene), EM-652, EM-800, EM-652-HCl(acolbifene, EM-1538), 4-hydroxy-Tamoxifen, 4-hydroxy-Toremifene,Droloxifene, LY 335563, GW-5638, Idoxifene, Levormeloxifene, Iproxifen(TAT-59), Ospemifene (FC 1271), Fispemifene, Centchroman, CHF 4227, LY2066948, LY 2120310, Sivifene, SR 16234, Clomiphene, Enclomiphene,Zuclomiphene, GW 7603, BL 3040, SRI 16158, SR 16157, SRI 16137, SR16137, Rad 1901,(+)-3-(4-hydroxyphenyl)-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-4-(trifluoromethyl)-2H-1-benzopyran-7-ol,Femarelle, Nafoxidine and Endoxifen.

It is preferred that the antiestrogen is selected from the groupcomprising of Faslodex (ICI 182780, fulvestrant,7α-[9-(4,4,5,5,5-pentafluoro-pentylsulphinyl)nonyl]oestra-1,3,5(10)-triene-3,17β-diol),ICI 164384, CH 4893237, ZK 246965 and SH 646.

It is preferred that the selective estrogen receptor modulator has oneof the following formulae selected from the group comprising of:

wherein R₁ and R₂ are independently hydrogen, hydroxyl, halogen, C₁-C₆alkyl or a moiety which is converted to hydroxyl in vivo;wherein Z is either absent or selected from the group consisting of—CH₂—, —O—, —S— and —NR₃— (R₃ being hydrogen or C₁-C₆ alkyl);wherein the R₁₀₀ is a bivalent moiety which distances L from the B-ringby 4-10 intervening atoms;wherein L is a bivalent or trivalent moiety selected from the group of—SO—, —CON<, —N<, and —SON<;wherein G₁ is selected from the group consisting of hydrogen, a C₁ to C₅hydrocarbon, a bivalent moiety which in combination with G₂ and L is a5- to 7-membered heterocyclic ring, and halo or unsaturated derivativesof the foregoing;wherein G₂ is either absent or selected from the group consisting ofhydrogen, a to C₅ hydrocarbon, a bivalent moiety which in combinationwith G₁ and L is a 5- to 7-membered heterocyclic ring, and halo orunsaturated derivatives of the foregoing;wherein G₃ is selected from the group consisting of hydrogen, methyl,ethyl and trifluoromethyl;

or a pharmaceutically acceptable salt thereof,wherein D is —OCH₂CH₂N(R₃)R₄ (R₃ and R₄ either being independentlyselected from the group consisting of C₁-C₄ alkyl, or R₃, R₄ and thenitrogen atom to which they are bound, together being a ring structureselected from the group consisting of pyrrolidinyl,2,2-dimethylpyrrolidinyl, 2-methylpyrrolidinyl, piperidino,hexamethyleneimino, and morpholino);wherein R₁ and R₂ are independently selected from the group consistingof: hydrogen, hydroxyl, halogen, C₁-C₆ alkyl, and a moiety converted invivo to hydroxyl;wherein G₃ is selected from the group consisting of hydrogen, methyl,ethyl and trifluoromethyl;

or a pharmaceutically acceptable salt thereof;wherein a benzopyran compound which is an optically active compoundhaving an absolute configuration S on carbon 2;wherein R₁ and R₂ are independently selected from the group consistingof hydroxyl, halogen, C₁-C₆ alkyl, and a moiety convertible in vivo tohydroxyl;wherein R³ is a species selected from the group consisting of saturated,unsaturated or substituted pyrrolidinyl, saturated, unsaturated orsubstituted piperidino, saturated, unsaturated or substitutedpiperidinyl, saturated, unsaturated or substituted morpholino,nitrogen-containing cyclic moiety, nitrogen-containing polycyclicmoiety, and NRaRb (Ra and Rb being independently hydrogen, straight orbranched C₁-C₆ alkyl, straight or branched C₂-C₆ alkenyl, or straight orbranched C₂-C₆ alkynyl);wherein a salt of an acid selected from the group consisting of aceticacid, adipic acid, benzenesulfonic acid, benzoic acid, camphorsulfonicacid, citric acid, fumaric acid, hydroiodic acid, hydrobromic acid,hydrochloric acid, hydrochlorothiazide acid, hydroxy-naphthoic acid,lactic acid, maleic acid, methanesulfonic acid, methylsulfuric acid,1,5-naphthalenedisulfonic acid, nitric acid, palmitic acid, pivalicacid, phosphoric acid, propionic acid, succinic acid, sulfuric acid,tartaric acid, terephthalic acid, p-toluenesulfonic acid, and valericacid.

In another embodiment, the invention provides a method which furthercomprising administering as part of a combination therapy, atherapeutically effective amount of human chorionic gonadotropin.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising:

-   -   a) a pharmaceutically acceptable excipient, diluent or carrier;    -   b) a therapeutically effective amount of at least one sex        steroid precursor or prodrug thereof; and    -   c) a therapeutically effective amount of at least one SERM,        antiestrogen or prodrug.

In another embodiment, the invention provides a pill, a tablet, acapsule, a gel, a cream, an ovule, a rectal suppository, or an injectioncomprising:

-   -   a) a pharmaceutically acceptable excipient, diluent or carrier;    -   b) a therapeutically effective amount of at least one sex        steroid precursor or prodrug thereof; and    -   c) a therapeutically effective amount of at least one SERM,        antiestrogen or prodrug.

In another embodiment, the invention provides a kit comprising a firstcontainer containing a pharmaceutical formulation comprising atherapeutically effective amount of at least one sex steroid precursoror a prodrug thereof; and said kit further comprising a second containercontaining a pharmaceutical formulation comprising a therapeuticallyeffective amount of at least one SERM, antiestrogen or prodrug as partof combination therapy.

In another embodiment, the invention pertains to a method of preventing,reducing or eliminating the incidence of male androgen deficiencysymptoms or diseases including male hypogonadism-associated symptoms anddiseases by increasing levels of a sex steroid precursor selected fromthe group consisting of dehydroepiandrosterone (DHEA),dehydroepiandrosterone-sulfate (DHEA-S),androst-5-ene-313,1713-diol(5-diol) and 4-androstene-3,17-dione in apatient in need of said prevention, reduction or elimination of theincidence, and further comprising administering to said patient atherapeutically effective amount of at least one SERM, antiestrogen orprodrug as part of combination therapy.

In another embodiment, the invention pertains to a method of preventing,reducing or eliminating the incidence of male androgen deficiencysymptoms or diseases including male hypogonadism-associated symptoms anddiseases by increasing levels of circulating testicular testosterone bythe action of SERM or antiestrogen in a patient in need of saidprevention, reduction or elimination of the incidence, and furthercomprising administering to said patient a therapeutically effectiveamount of at least one sex steroid precursor or prodrug as part ofcombination therapy.

In another embodiment, the invention provides a method of preventing,reducing or eliminating the incidence of male androgen deficiencysymptoms or diseases including male hypogonadism-associated symptoms anddiseases by increasing the levels of circulating androgen metabolitesconsisting of androsterone glucuronide (ADT-G),androstane-3α,17β-diol-3-glucuronide (3α-diol-3G) andandrostane-3α,17β-diol-17-glucuronide (3α-diol-17G), said methodcomprising administering to male patient in need of said prevention,reduction or elimination, a therapeutically effective amount of a sexsteroid precursor or prodrug thereof in association with atherapeutically effective amount of a selective estrogen receptormodulator or an antiestrogen or prodrug of either.

As used herein, “Pure SERM” means that the SERM does not have anyestrogenic activity in breast or uterine tissue at physiological orpharmacological concentrations.

In another embodiment, the invention provides a kit comprising a firstcontainer containing a therapeutically effective amount of at least oneprecursor of sex steroids and further comprising a second containercontaining a therapeutically effective amount of at least one SERM.

In another embodiment, the invention provides, in one container, apharmaceutical composition comprising:

-   -   a) a pharmaceutically acceptable excipient, diluent or carrier;    -   b) a therapeutically effective amount of at least one precursor        of sex steroids; and    -   c) a therapeutically effective amount of at least one SERM.

In another embodiment, the invention provides a method of preventing,reducing or eliminating the incidence of male androgen deficiencysymptoms or diseases including male hypogonadism-associated symptoms anddiseases, said method comprising administering to male patient in needof said prevention, reduction or elimination, a therapeuticallyeffective amount of a sex steroid precursor or prodrug thereof inassociation with a therapeutically effective amount of a selectiveestrogen receptor modulator or an antiestrogen or prodrug of either,wherein the selective estrogen receptor modulator or antiestrogenstimulates LH secretion which increases the level of circulatingtestosterone.

In another embodiment, the invention provides a pharmaceuticalcomposition for preventing, reducing or eliminating the incidence ofmale androgen deficiency symptoms or diseases including malehypogonadism-associated symptoms and diseases comprising:

-   -   a) a pharmaceutically acceptable excipient, diluent or carrier;    -   b) at least one sex steroid precursor or prodrug thereof; and    -   c) at least one selective estrogen receptor modulator or an        antiestrogen or prodrug of either;        wherein said pharmaceutical composition is provided in packaging        that directs use of said composition for prevention, reduction        or elimination of at least one male androgen deficiency symptom        or disease.

In another embodiment, the invention provides a kit for preventing,reducing or eliminating the incidence of male androgen deficiencysymptoms or diseases including male hypogonadism-associated symptoms anddiseases, comprising (i) a first container having therein at least onesex steroid precursor or a prodrug thereof; (ii) a second containerhaving therein at least one selective estrogen receptor modulator, or anantiestrogen or prodrug of either of the foregoing; and (iii)instructions for using the kit for the prevention, reduction orelimination of at least one male androgen deficiency symptom or disease.

It is preferred that the sex steroid precursor is dehydroepiandrosteroneand the selective estrogen receptor modulator is acolbifene.

As used herein, compounds administered to a patient “in associationwith” other compounds are administered sufficiently close toadministration of said other compound that a patient obtains thephysiological effects of both compounds simultaneously, even though thecompounds were not administered in close time proximity. When compoundsare administered as part of a combination therapy they are administeredin association with each other. Preferred SERM (acolbifene) discussedherein is preferably used in combination with preferred sex steroidprecursors dehydroepiandrosterone, dehydroepiandrosterone-sulfate,androst-5-ene-3β,17β-diol or 4-androstene-3,17-dione, especiallydehydroepiandrosterone.

The applicant believes that the addition of a precursor of sex steroidsto acolbifene treatment will increase intracellular levels oftestosterone (as well demonstrated in patients with prostate cancerwhere intracellular androgens, especially dihydrotestosterone, is comingfrom endogenous DHEA (Labrie, Dupont et al. 1985; Labrie, Cusan et al.2009; Labrie 2011)).

As used herein, a SERM is a compound that functions as an estrogenreceptor antagonist (antiestrogen) in breast tissue, yet providesestrogenic or estrogen-like effect on bone tissue and on serumcholesterol levels (i.e. by reducing serum cholesterol). Non-steroidalcompounds that function as estrogen receptor antagonists in vitro or inhuman or rat breast tissue (especially if the compound acts as anantiestrogen on human breast cancer cells) is likely to function as aSERM. Conversely, steroidal antiestrogens tend not to function as SERMsbecause they tend not to display any beneficial effect on serumcholesterol. Non-steroidal antiestrogens we have tested and found tofunction as SERMs include EM-800, EM-652.HCl, raloxifene, tamoxifen,4-hydroxy-tamoxifen, toremifene, 4-hydroxy-toremifene, droloxifene, LY353 381, LY 335 563, GW-5638, lasofoxifene, bazedoxifene (TSE 424;WAY-TSE 424; WAY 140424;1-[[4[2-(hexahydro-1H-azepin-1-yl)ethoxy]phenyl]methyl]-2-(4-hydroxyphenyl)-3methyl-1H-indol-5-ol), pipendoxifene (ERA 923;2-(4-hydroxyphenyl)-3-methyl-1-[[4-[2-(1-piperidinyl)ethoxy]phenyl]methyl]-1H-indol-5-ol)ospemifene and idoxifene, but are not limited to these compounds.

But we have found also that all SERMs do not react in the same mannerand may be divided into two subclasses: “pure SERMs” and “mixed SERMs”.Thus, some SERMs like EM-800 and EM-652.HCl do not have any estrogenicactivity in breast and endometrial tissues at physiological orpharmacological concentrations and have hypocholesterolemic andhypotriglyceridemic effects in the rat. These SERMS may be called “pureSERMs”. The ideal SERM is a pure SERM of the type EM-652.HCl because ofits potent and pure antiestrogenic activity in the mammary gland.Others, like raloxifene, tamoxifen, droloxifene, 4-hydroxy-tamoxifen(1-(4-dimethylaminoethoxyphenyl)-1-(4-hydroxylphenyl)-2-phenyl-but-1-ene),toremifene,4-hydroxy-toremifene[(Z)-(2)-2-[4-(4-chloro-1-(4-hydroxyphenyl)-2-phenyl-1-butenyl)phenoxyl]-N,N-dimethylethanamine),LY 353 381, LY 335 563, GW-5638, lasofoxifene, idoxifene, bazedoxifeneand ospemifene have some estrogenic activities in the breast andendometrium. This second series of SERMs may be called “mixed SERMs”.The unwanted estrogenic activities of these “mixed SERMs” may beinhibited by addition of pure “SERMs” as shown in FIGS. 2 and 3 in vitrotests and in FIG. 4 in an in vivo test of breast cancer. Since humanbreast carcinoma xenografts in nude mice are the closest available modelof human breast cancer, we have thus compared the effect of EM-800 andtamoxifen alone and in combination on the growth of ZR-75-1 breastcancer xenografts in nude mice.

In one embodiment, the invention uses selective estrogen receptormodulators of the following molecular structure

wherein R1 and R2 are independently hydrogen, hydroxyl or a moiety whichis converted to hydroxyl in vivo, and n=1 or 2.

The applicant believes that it is very important that SERMs of theinvention act as pure antiestrogens in breast because SERMs have tocounteract potential side-effects of estrogens, particularly thoseformed from the exogenous precursors of sex steroids which can increasethe proliferation of this tissue. Particularly, the applicant believesthat benzopyran derivatives of the invention having the absoluteconfiguration 2S at position 2 is more suitable than its racemicmixture. Thus, in U.S. Pat. No. 6,060,503, optically active benzopyranantiestrogens having 2S configuration are disclosed to treatestrogen-exacerbated breast and endometrial cancer and these compoundsare shown to be significantly more efficient than racemic mixtures (SeeFIGS. 1-5 of U.S. Pat. No. 6,060,503).

The enantiomer of 2S configuration being difficult to be industriallyobtained as a pure state, the applicant believes that less than 10%,preferably less than 5% and more preferably less than 2% by weight ofcontamination by the 2R enantiomer is preferred.

Prodrug forms of active pharmaceutical ingredient are well known in theart. See, e.g. H. Bundgaard “5. Design and Application of Prodrugs” (InA textbook of Drug Design and Development. Edited by P.Krogsgaard-Larsen and H. Bundgaard; Harwood Academic Publishers GmbH,Chur, Switzerland, 1991), the contents of which are incorporated hereinby reference. In particular, see page 114 defining prodrug: a prodrug isa pharmacologically inactive derivative of a parent drug molecule thatrequires spontaneous or enzymatic transformation within the body inorder to release the active drug, and that has improved deliveryproperties over the parent drug molecule. In the present application,the prodrugs of sex steroid precursor are derivatives of the 3- and/or17-hydroxyl group(s) and/or 3- and/or 17-ketone group(s), and theprodrugs of selective estrogen receptor modulators and antiestrogens arederivatives of the hydroxyl group. The prodrug forms of the hydroxylgroup are esters, carbonate esters, phosphate esters, ethers, andα-acyloxyalkyl ethers, and the prodrug forms of the ketone group areketals, imines, enol esters, oxazolidines and thiazolidines but notlimited by these examples (see page 154). The previous-cited SERM EM-800(diester derivative, dipivaloate) is a prodrug of EM-652 (Gauthier,Caron et al. 1997).

Serum testosterone is higher in the morning and decreases to a minimumconcentration after sleep (Trenell, Marshall et al. 2007). Serumtestosterone should be monitored (with the judgment of the treatingphysician concerning its frequency) at months 1 and 2 of treatment andthen every 3 months to assure proper increases in serum testosterone.Similar measurements should be made for DHEA. Serum DHEA also follows acircadian rythm being lowest in the morning. For proper comparison, itis preferable to measure serum testosterone and DHEA at the same time ofthe day at different treatment time intervals, i.e., at month 1 and 2and then every 3 months.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of thehypothalamo-pituitary-testicular and hypothalamo-pituitary-adrenal axes.GnRH, gonadotropin-releasing hormone; CRH, corticotropin-releasinghormone; LH, luteinizing hormone; ACTH, adrenocorticotropin; DHEA,dehydroepiandrosterone; E₂, estradiol; DHT, dihydrotestosterone; Testo,testosterone.

FIG. 2 shows the effect of increasing concentrations of EM-800 (prodrugof acolbifene, free salt), (Z)-4-OH-tamoxifen, (Z)-4-OH-toremifene andraloxifene on alkaline phosphatase activity in human endometrial cancerIshikawa cells. Alkaline phosphatase activity was measured after a 5-dayexposure to increasing concentrations of indicated compounds in thepresence or absence of 1.0 nM E₂. The data are expressed as themeans±SEM of four wells. When SEM overlaps with the symbol used, onlythe symbol is shown (Simard, Sanchez et al. 1997).

FIG. 3 shows the blockade of the stimulatory effect of(Z)-4-OH-tamoxifen, (Z)-4-OH-toremifene, droloxifene and raloxifene onalkaline phosphatase activity by the antiestrogen EM-800 (prodrug ofacolbifene, free salt) in human Ishikawa (endometrial) carcinoma cells.Alkaline phosphatase activity was measured after a 5-day exposure to 3or 10 nM of the indicated compounds in the presence or absence of 30 or100 nM EM-800. The data are expressed as the means±SD of eight wellswith the exception of the control groups were data are obtained from 16wells (Simard, Sanchez et al. 1997).

FIG. 4 shows that the stimulatory effect of tamoxifen on the growth ofhuman breast cancer ZR-75-1 xenografts is completely blocked bysimultaneous administration of EM-652.HCl (acolbifene). Acolbifene, byitself, in agreement with its pure antiestrogenic activity has no effecton tumor growth in the absence of tamoxifen.

FIG. 5. Schematic representation of the adrenal and intracrinesteroidogenic pathways, DHEA, dehydroepiandrosterone; DHEA-S,DHEA-sulphate; DHT, dihydrotestosterone; HSD, hydroxysteroiddehydrogenase.

FIG. 6. Comparison of the serum concentrations of testosterone (A),total androgenic pool (sum of ADT-G, 3α-diol-3G and 3α-diol-17G) (B) andE₁S (C) in castrated 69-80-year-old men (n=34) and intact 55-65-year-oldpostmenopausal women (n=377) (Labrie, Bélanger et al. 2006; Labrie,Cusan et al. 2009).

FIG. 7 shows the effect of 12-month treatment withdehydroepiandrosterone (DHEA) alone or in combination with Flutamide orEM-800 (prodrug of acolbifene, free salt) on trabecular bone volume inovariectomized rats. Intact animals are added as additional controls.Data are presented as mean±SEM**p<0.01 versus OVX Control.

FIG. 8 shows the effect of 12-month treatment withdehydroepiandrosterone (DHEA) alone or in combination with Flutamide orEM-800 (prodrug of acolbifene, free salt) on trabecular number inovariectomized rats. Intact animals are added as additional controls.Data are presented as mean±SEM**p<0.01 versus OVX Control.

FIG. 9 shows proximal tibia metaphyses from intact control (A),ovariectomized control (B), and ovariectomized rats treated with DHEAalone (C) or in combination with Flutamide (D) or EM-800 (prodrug ofacolbifene, free salt) (E). Note the reduced amount of trabecular bone(T) in ovariectomized control animals (B), and the significant increasein trabecular bone volume (T) induced after DHEA administration (C). Theaddition of Flutamide to DHEA partially blocked the effect of DHEA onthe trabecular bone volume (D), whereas the combination of DHEA andEM-800 (prodrug of acolbifene, free salt) provided complete protectionagainst the ovariectomy-associated bone loss. Modified trichromeMasson-Goldner, magn.x80. T: Trabeculae, GP: Growth Plate.

FIG. 10 shows the effect of treatment with DHEA (10 mg, percutaneously,once daily) or EM-800 (prodrug of acolbifene, free salt) (75 μg, orally,once daily) alone or in combination for 9 months on serum triglycerides(A) and cholesterol (B) levels in the rat. Data are expressed as themeans±SEM. **: p<0.01 experimental versus respective control.

FIG. 11 shows the effect of 37-week treatment with increasing doses(0.01, 0.03, 0.1, 0.3, and 1 mg/kg) of EM-800 (prodrug of acolbifene,free salt) or raloxifene administered on total serum cholesterol levelsin the ovariectomized rat. Comparison is made with intact rats andovariectomized animals bearing an implant of 17β-estradiol (E₂); **p<0.01, experimental versus OVX control rats.

FIG. 12: Schematic representation of the role of testicular and adrenalsources of sex steroids in men and the effect of adding acolbifene tocounteract the inhibitory effect of estrogens at thehypothalamo-pituitary level on the secretion of LH. ACTH,adrenocorticotropin; CRH, corticotropin-releasing hormone; DHEA,dehydroepiandrosterone; DHT, dihydrotestosterone; E₂, 17β-estradiol; LH,luteinizing hormone; GnRH, gonadotropin-releasing hormone.

FIG. 13: Male cynomolgus monkeys were dosed orally with 2.5, 10 or 40 mgacolbifene/day for 13 weeks. Control monkeys received vehicle alone(0.4% methylcellulose). End of study serum testosterone concentrationswere determined using a validated gas chromatography mass spectrometricassay. Results are expressed as the mean±SEM of 4 monkeys per group. Pvalues (versus control) were calculated using a two-sided t testassuming equality of variances.

FIG. 14: Male cynomolgus monkeys were dosed orally with 3.13, 12.5 or 50mg EM-800 (prodrug of acolbifene, free salt)/day for 52 weeks. Controlmonkeys received vehicle alone (0.4% methylcellulose). End of studyserum testosterone concentrations were determined using a validated gaschromatography mass spectrometric assay. Results are expressed as themean±SEM of 5 (EM-800 study) monkeys per group. P values (versuscontrol) were calculated using a two-sided t test assuming equality ofvariances.

FIG. 15 shows the effects of antiestrogens on ZR-75-1 tumor growth.Effect of treatment with the antiestrogens tamoxifen, EM-652.HCl(acolbifene) and the combination of tamoxifen and EM-652.HCl for 161days, on the growth of human ZR-75-1 breast tumors in ovariectomizednude mice. Tumor size is expressed as the percentage of initial tumorarea (Day 1=100%). Data is expressed as means±SEM (n=18-30tumors/group); ##p<0.01 vs EM-652.HCl (acolbifene); **p<0.01 vs OVX.Antiestrogens were administered orally once daily at the dose of 200μg/mouse in absence of estrogen stimulation.

FIG. 16 Effect on uterine weight of increasing daily doses of theantiestrogens CS-115-1 (EM-343) and EM-762 administered orally orpercutaneously by application on the skin for 9 days to ovariectomizedmice simultaneously treated by twice daily subcutaneous injection ofestrone.

FIG. 17 shows the effect on uterine weight of increasing concentrationsof EM-652.HCl (acolbifene), lasofoxifene (free base; active and inactiveenantiomers) and raloxifene administered orally for 9 days toovariectomized mice simultaneously treated with estrone. *p<0.05,**p<0.01 versus E₁-treated control.

FIG. 18 shows the effect on uterine weight of 1 μg and 10 μg ofEM-652.HCl (acolbifene), lasofoxifene (free base; active and inactiveenantiomers) and raloxifene administered orally for 9 days toovariectomized mice. **p<0.01 versus OVX control.

FIG. 19 shows the effects of antiestrogens on ZR-75-1 tumor growth.Effect of treatment with 7 antiestrogens for 161 days, onestrone-induced growth of human ZR-75-1 breast tumors in ovariectomizednude mice. Tumor size is expressed as the percentage of initial tumorarea (Day 1=100%). Data is expressed as means±SEM (n=18-30tumors/group); ## p<0.01 vs EM-652.HCl (acolbifene); ** p<0.01 vs OVX.Antiestrogens were administered orally once daily at the dose of 50μg/mouse under estrone stimulation obtained with subcutaneous 0.5-cmsilastic implants containing 1:25 ratio of estrone and cholesterol.

FIG. 20 shows the effects of antiestrogens on ZR-75-1 tumor growth.Effect of treatment with 7 antiestrogens for 161 days, on the growth ofhuman ZR-75-1 breast tumors in ovariectomized nude mice. Tumor size isexpressed as the percentage of initial tumor area (Day 1=100%). Date isexpressed as means±SEM (n=18-30 tumors/group); ## p<0.01 vs EM-652.HCl(acolbifene); **p<0.01 vs OVX. Antiestrogens were administered orallyonce daily at the dose of 100 μg/mouse in absence of estrogenstimulation.

FIG. 21 shows the effects of the combination of dehydroepiandrosteroneand the SERM acolbifene on various parameters. The addition ofacolbifene to dehydroepiandrosterone will treat or reduce the indicatednegative effects of low androgens.

DETAILED DESCRIPTION OF THE INVENTION Beneficial Effects of DHEA

We feel that the increased understanding of androgen and estrogenformation and action in peripheral target tissues called intracrinology(Labrie 1991; Labrie, Simard et al. 1992a; Labrie, Simard et al. 1992b;Labrie, Simard et al. 1994; Labrie, Durocher et al. 1995; Luu-The,Dufort et al. 1995; Labrie, Simard et al. 1996b; Labrie, Bélanger et al.1997a; Labrie, Bélanger et al. 1997b; Labrie, Diamond et al. 1997b;Labrie, Luu-The et al. 1997) as well as our recent observationsindicating the predominant role of androgens over that of estrogens inthe prevention of bone loss after ovariectomy in the rat (Martel, Sourlaet al. 1998) and the observation of a similar situation inpostmenopausal women (Labrie, Diamond et al. 1997a) have paved the wayfor a timely and potentially highly significant progress in the field ofsex steroid replacement therapy and aging. Such a possibility is wellsupported by our observations.

The present invention is thus based upon the recent progress achieved inour understanding of sex steroid physiology in men and women (Labrie1991; Labrie, Simard et al. 1992a; Labrie, Simard et al. 1992b; Labrie,Simard et al. 1994; Labrie, Durocher et al. 1995; Luu-The, Dufort et al.1995; Labrie, Simard et al. 1996b; Labrie, Bélanger et al. 1997a;Labrie, Bélanger et al. 1997b; Labrie, Diamond et al. 1997b; Labrie,Luu-The et al. 1997).

The pool of androgens in men decreases progressively from the age of 30years in parallel with the decrease in the serum concentration of DHEAand DHEA-S(Labrie, Bélanger et al. 1997b). Since serum DHEA isresponsible for up to 50% of the androgens present in peripheral tissues(Labrie, Dupont et al. 1985; Labrie, Cusan et al. 2009; Labrie 2010b;Labrie 2011) such a decrease of the biosynthesis of androgens from DHEAwith aging is likely to play an important role in the appearance of LOH(Late Onset Hypogonadism) and all the problems mentioned earlier relatedto low androgens.

DHEA, an Important Source of Peripheral Androgens Made by the IntracrineMechanisms in Men

Humans, with some other primates, are unique among animal species inhaving adrenals that secrete large amounts of the inactive precursorsteroids DHEA and DHEA-S, which are converted into potent androgensand/or estrogens in peripheral tissues. It is remarkable that man, inaddition to possessing very sophisticated endocrine and paracrinesystems, has largely invested in sex steroid formation in peripheraltissues (Labrie, Dupont et al. 1985; Labrie, Bélanger et al. 1988;Labrie 1991; Labrie, Bélanger et al. 1997a) (FIGS. 1,2 and 5).

In men, the 95% (or more) fall in serum testosterone induced bycastration and the clinical benefits of this partial elimination ofandrogens with advanced prostate cancer (Huggins and Hodges 1941) haveled to erroneously believe that castration eliminates 95% (or more) ofandrogens and that castration alone is an appropriate treatment forprostate cancer.

In men, the finding that 25-50% of androgens are left in the prostateafter castration (Labrie, Dupont et al. 1985; Bélanger, Bélanger et al.1989; Nishiyama, Hashimoto et al. 2004; Mostaqhel, Page et al. 2007)explains why the addition of a pure (non-steroidal) anti-androgen tocastration achieves a more complete blockade of androgens and has beenthe first treatment shown to prolong life in prostate cancer (Labrie,Dupont et al. 1982; Labrie, Dupont et al. 1985; Caubet, Tosteson et al.1997; Prostate Cancer Triallists' Collaborative Group 2000; Labrie,Bélanger et al. 2005). The androgens remaining at relatively high levelsafter castration also explain why combined androgen blockades or theblockade of the androgens of both testicular and adrenal origins atstart of treatment can provide cure for most patients when the treatmentis started at the localized stage of the cancer (Labrie, Candas et al.2002; Akaza 2006; Ueno Namiki et al. 2006), thus clearly demonstratingthe major role of extratesticular androgens or intracrinology in men.

Transformation of the adrenal precursor steroid DHEA into androgensand/or estrogens in peripheral target tissues depends upon the levels ofexpression of the various steroidogenic and metabolizing enzymes in eachcell of these tissues. This situation of a high secretion rate ofadrenal precursor sex steroids in men and women is thus completelydifferent from all animal models used in the laboratory (namely rats,mice, guinea pigs and all others except monkeys), where the secretion ofsex steroids takes place exclusively in the gonads (Labrie, Dupont etal. 1985; Labrie, Bélanger at al. 1988; Bélanger, Bélanger et al. 1989;Labrie, Bélanger et al. 1997a).

The androgens testosterone and DHT as well as E₂ made in peripheraltissues from DHEA of adrenal origin exert their action locally in thesame cells where their synthesis takes place (FIG. 5). Thissophisticated mechanism permits to maintain biologically active levelsof intracellular estrogens and/or androgens in specific tissues in needof these sex steroids while the same steroids leak in the blood at verylow levels, thus sparing the other tissues from a potentially negativeinfluence. Following their cell-specific local formation and immediateavailability for local intracellular action, testosterone and DHT (themost active natural androgen) and E₂ are inactivated and transformed inthe same cells into water-soluble glucuronide or sulphate derivativeswhich can then diffuse quantitatively into the general circulation wherethey can be measured by mass spectrometry (Labrie, Bélanger et al. 2006)before their elimination by the kidneys.

It should also be noted that the importance of the intracrine formationof androgens and estrogens extends to non-malignant diseases such asacne, seborrhoea, hirsutism and androgenic alopecia as well as toosteoporosis and vulvovaginal atrophy (Cusan, Dupont et al. 1994;Labrie, Bélanger et al. 1997a; Labrie, Archer et al. 2009b; Labrie,Archer et al. 2009a; Labrie, Archer et al. 2009c). Practically alltissues possess, at various levels, a battery of steroidogenic enzymesthat can transform DHEA. Each tissue, however, possesses a highlytissue-specific set of steroidogenic and steroid-inactivating enzymeswhich require experimentation to be known.

While the serum levels of testosterone are reduced by 97.4% followingcastration in 69-80-year-old men (Labrie, Cusan et al. 2009), the sum ofthe metabolites of androgens, the only accurate and valid parameter oftotal androgenic activity measurable in the circulation (Labrie,Bélanger et al. 2006), is only reduced by 58.9% (Labrie, Cusan et al.2009), thus indicating that a very important proportion (41.1%) ofandrogens remains in men after complete elimination of testicularandrogens. Such data are in close agreement with the concentration ofintraprostatic DHT that shows that, on average, 39% of DHT is left inthe prostate after castration in various studies, namely 45% (Labrie,Dupont et al. 1985), 51% (Bélanger, Brochu et al. 1986), 25% (Nishiyama,Hashimoto at al. 2004) and 35% (Mostaghel, Page et al. 2007) (see FIG. 4in (Labrie 2010b)).

With the knowledge of the major importance of androgens of adrenalorigin in men, it is of interest to compare the data mentioned above formen with the serum levels of the same steroids measured in intactpostmenopausal women. As can be seen in FIGS. 6A and 6B, the serumlevels of testosterone and of the total androgen metabolites are almostsuperimposable in castrated men and postmenopausal women of comparableage. Most interestingly, it can also be seen that the serum levels ofestrone sulphate (E₁S) are also comparable (FIG. 6C). It could also beseen that the serum levels of E₁ and E₂ are also comparable, thusindicating that similar amounts of estrogens of adrenal origin are foundin both men and women (Labrie, Cusan et al. 2009).

The above-summarized data show that ˜40% of androgens are made inperipheral tissues in the absence of testicles in 69-80-year-old men.Since serum DHEA decreases markedly with age starting in the thirties(Labrie, Dupont et al. 1985), and testicular androgen secretiondecreases only slightly, it is most likely that androgens of adrenalorigin have an even greater relative and absolute importance at youngerages.

As mentioned above, the local synthesis and action of sex steroids inperipheral target tissues has been called intracrinology (Labrie,Bélanger et al. 1988; Labrie 1991). Recent and rapid progress in thisarea has been made possible by the elucidation of the structure of mostof the tissue-specific genes that encode the steroidogenic enzymesresponsible for the transformation of DHEA-S and DHEA into androgensand/or estrogens locally in peripheral tissues (Labrie, Simard et al.1992a; Labrie, Sugimoto et al. 1992; Labrie, Durocher et al. 1995;Luu-The, Zhang et al. 1995; Labrie, Simard et al. 1996a; Labrie, Luu-Theet al. 1997) (FIG. 5).

The major importance of DHEA and DHEA-S in human sex steroid physiologyis illustrated by the estimate that up to 50% of total androgens inadult men derive from these adrenal precursor steroids (Labrie, Dupontet al. 1985; Bélanger, Brochu et al. 1986; Labrie, Bélanger et al.1993).

Concerning the breast, DHEA is known to prevent the development (LuoSourla et al. 1997) and to inhibit the growth (Li, Yan et al. 1993) ofdimethylbenz(a)anthracene mammary tumors in the rat. DHEA, in addition,inhibits the growth of human breast cancer xenografts in nude mice (Seeexample 1 and (Couillard, Labrie et al. 1998). Thus, contrary toestrogens and progestins which exert stimulatory effects, DHEA isexpected to inhibit both the development and the growth of breast cancerin women.

As well demonstrated in our previous studies, supplementation withphysiological amounts of exogenous DHEA permits the biosynthesis ofandrogens and estrogens only in the appropriate target tissues whichcontain the specific steroidogenic enzymes. The active androgens andestrogens thus synthesized remain in the cells of origin and very littleleakage occurs into the circulation.

In fact, the most striking effects of DHEA administration are on thecirculating levels of the glucuronide derivatives of the metabolites ofDHT, namely ADT-G and 3α-diol-G, these metabolites being producedlocally in the peripheral intracrine tissues which possess theappropriate steroidogenic enzymes to synthesize DHT from the adrenalprecursors DHEA and DHEA-S and, thereafter, to further metabolize DHTinto inactive conjugates (Labrie 1991; Labrie, Simard et al. 1996a).This local biosynthesis and action of androgens in target tissueseliminates the exposure of other tissues to androgens and thus minimizesthe risks of undesirable masculinizing or other androgen-related sideeffects. The same applies to estrogens although we feel that a reliableparameter of total estrogen secretion (comparable to the glucuronidesfor androgens) is not yet available.

DHEA, Muscle and Lean Body Mass

Since 40-50% of androgens in 60-70-year-old men originate from adrenalDHEA (Labrie, Cusan et al. 2009), it is reasonable to believe thatadrenal DHEA has an importance comparable to testicular testosterone inthe control of muscle mass and strength in men.

There is no doubt that androgens play the predominant role in musclegrowth, development and function. Androgens are well known to increasemuscle mass in normal men (Bhasin, Storer et al. 1996; Bhasin, Woodhouseet al. 2001), this effect being related to the ban of androgens by theInternational Olympic Committee. In fact, the major form of sportsdoping remains androgenicanabolic abuse. At suitable doses, exogenousandrogens enhance muscle mass and strength in all men and women athletes(Handelsman 2006). As a result, since the early 1970s, exogenousandrogens have been banned for men and women in sports.

The marked decline in serum DHEA in aging women and men has led to thesuggestion that a series of changes associated with aging, includingloss of muscle mass and strength, may be due to declining DHEA with age(Labrie, Bélanger et al. 1998; Lamberts 2003). The beneficial effects ofDHEA in rodents on body composition are well known (Tagliaferro, Davisat al. 1986; Han, Hansen et al. 1998). Several age-related changesobserved in men, especially loss of muscle and bone mass, as well assexual function and increase in fat mass are similar to those observedin androgen deficiency (Matsumoto 2002; Morley and Perry 2003).

Based on cross-sectional data, maximal muscle strength at the age of 70years is 30-50% of peak muscle strength found at the age of 30 years(Murray and Pitt 1985; Kallman, Plato et al. 1990). The age associatedmuscle strength loss seems to be correlated with a reducedcross-sectional area of the muscles (Larsson, Grimby at al. 1979;Kallman, Plato et al. 1990). Age-related sarcopenia increases the riskof falls, fractures, disability and life-threatening complications(Evans 1997; Frontera, Hughes et al. 2000; Melton, Khosla et al. 2000;Hughes, Frontera et al. 2002; Iannuzzi-Sucich, Prestwood et al. 2002).

Following studies where apparently too low doses of testosterone wereused (Elashoff, Jacknow et al. 1991), a series of recent studies haveunequivocally demonstrated a dose-response stimulatory effect ofandrogens on muscle size and strength (Bhasin, Storer et al. 1996;Bhasin, Storer et al. 1997; Bross, Casaburi et al. 1998; Bhasin,Woodhouse et al. 2001; Storer, Magliano et al. 2003; Bhasin, Woodhouseet al. 2005) have compared the efficacy of increasing doses oftestosterone on androgen-sensitive parameters in 60-75-year-old and19-35-year-old men. All men were treated with a GnRH agonist toeliminate endogenous and variable levels of testicular androgens. Theweekly doses of testosterone enanthate were 25, 50, 125, 300 and 600 mgfor 20 weeks. The effects observed in both young and old men were doserelated. The increases in fat-free mass and muscle strength werecorrelated with the testosterone dose and were not different in old andyoung men. The best tolerance was achieved with the 125 mg dose, a dosegiving high normal serum testosterone levels, low levels of adverseeffects and an increase in fat-free mass and muscle strength (Bhasin,Woodhouse et al. 2005). The effects of androgens on the muscle are wellrecognized in hypogonadal men (Bhasin, Storer et al. 1997; Snyder,Peachey et al. 2000) and men receiving glucocorticoid therapy (Crawford,Liu et al. 2003).

In a study of 558 men aged 20-95 years, serum DHEA-S was found to be anindependent predictor of muscle strength and mass in men aged 60-79years (Valenti, Denti et al. 2004). These results are in agreement withanother study showing a correlation between serum DHEA-S and musclepower (Kostka, Arsac et al. 2000; Bonnefoy, Patricot et al. 2002).

The administration of a daily dose of 50 or 100 mg DHEA for 6 or 12months, respectively, improved knee extension strength in older men(Yen, Morales et al. 1995). No significant effect, however, was foundfollowing the administration of DHEA in 60-80-year-old women but thenumber of subjects was small. Muscle mass increase following DHEAadministration has been observed by (Yen, Morales et al. 1995; Diamond,Cusan et al. 1996; Morales, Haubrich et al. 1998; Gebre-Medhin, Husebyeet al. 2000; Villareal, Holloszy et al. 2000; Gordon, Grace et al. 2002;Johannsson, Burman et al. 2002) while others found no significant effect(Yen, Morales et al. 1995; Callies, Fassnacht et al. 2001; Percheron,Hogrel et al. 2003) in women.

Lean body mass has been reported to be increased by DHEA treatment(Diamond, Cusan et al. 1996; Morales, Haubrich et al. 1998;Gebre-Medhin, Husebye et al. 2000; Villareal, Holloszy et al. 2000;Nair, Rizza et al. 2006; Gurnell, Hunt et al. 2008).

Postural imbalance and falls are increasingly associated with hipfractures during aging (Cummings and Nevitt 1989). In fact, it isestimated that 80% of fractures in the elderly occur in the absence ofperipheral osteoporosis (Siris, Chen et al. 2004) Such data stress themajor importance of preventing falls in older adults by maintainingmuscle mass and strength (Chang, Morton et al. 2004). A large proportionof fractures thus result from falls due to loss of muscle mass andstrength which should be preventable, up to an unknown extent, byappropriate DHEA replacement.

Role of Androgens and Estrogens in Bone Physiology

A predominant role of androgens on bone physiology is well documented(Labrie, Diamond et al. 1997b; Martel, Sourla et al. 1998) In fact, bothtestosterone and DHT increased the transcription of a (I) procollagenmRNA in osteoblast-like osteosarcoma cells (Benz, Haussler et al. 1991).Treatment with DHT has also been shown to stimulate endochondral bonedevelopment in the orchiectomized rat (Kapur and Reddi 1989). Moreover,bone mineral density measured in the lumbar spine, femoral trochanterand total body was increased more by estrogen+testosterone implants thanby E₂ alone over a 24-month treatment period in postmenopausal women(Davis, McCloud et al. 1995).

Moreover, in established osteoporosis, anabolic steroids have beenreported to help prevent bone loss (Hennernan and Wallach 1957).Similarly, subcutaneous E₂ and testosterone implants have been found tobe more efficient than oral estrogen in preventing osteoporosis inpostmenopausal women (Savvas, Studd et al. 1988). Although thedifference observed in that study has been attributed to the differentroutes of administration of the estrogen, the cause of the differencecould well be the action of testosterone. As index of increased boneformation, an increase in serum osteocalcin, a marker of bone formationhas been found in postmenopausal women receiving methyltestosterone plusestrogen, compared with estrogen alone (Raisz, Wiita et al. 1996). Asimilar stimulatory effect on serum osteocalcin has been observedfollowing treatment of postmenopausal women with percutaneous DHEA for12 months (Labrie, Diamond et al. 1997a). Moreover, androgen therapy, asobserved with nandrolone decanoate, has been found to increase vertebralbone mineral density in postmenopausal women (Need, Horowitz et al.1989). Although androgens are gaining increasing support due to theirunique actions in postmenopausal women, virilizing effects are observedwith the use of testosterone (Burger, Hailes et al. 1984; Studd, Collinset al. 1987).

We have shown that DHEA exerts beneficial effects on bone in both thefemale rat (Luo, Sourla et al. 1997), and postmenopausal women (Labrie,Diamond et al. 1997a). Thus, in intact female rats, treatment with DHEAincreases bone mineral density (BMD) of total skeleton, lumbar spine andfemur (Luo, Sourla et al. 1997) (FIGS. 7, 8 and 9).

That the SERMs raloxifene and toremifene increase bone mineral densityhas been demonstrated (Smith 2006). Clomiphene citrate has shownpositive results on serum testosterone and symptoms/signs ofhypogonadism (Shabsiqh, Kang et al. 2005; Whitten, Nanqia et al. 2006).

DHEA and Abdominal Obesity

Abdominal obesity is associated with an increased risk of insulinresistance, type 2 diabetes and atherosclerosis (Shimokata, Tobin et al.1989; Cefalu, Wang et al. 1995; Ferrannini, Natali et al. 1997; Kopelman2000). Among other factors, hormonal changes, especially the decliningsecretion of DHEA and DHEA-S by the adrenals is thought to be a factorinvolved (Tchernof, Labrie et al. 1996). In rat and mouse models, DHEAadministration reduces visceral fat accumulation in diet-induced (Yen,Allan et al. 1977; Cleary and Zisk 1986; Mohan, Ihnen et al. 1990;Hansen, Han et al. 1997) obesity. A beneficial effect of DHEA has alsobeen observed on the decrease in insulin resistance that occurs with age(Han, Hansen et al. 1998).

In a study performed in postmenopausal women who received a DHEA creamfor 12 months, we have found that insulin resistance was decreased whilesubcutaneous fat at the level of the thigh was also decreased (Diamond,Cusan et al. 1996). Moreover, the daily administration of 50 mg DHEA for6 months in 65 to 78-year-old men and women decreased abdominal visceralfat by 10.2% in women and 7.4% in men (Villareal and Holloszy 2004). Inthe same study, abdominal subcutaneous fat was decreased by 6% in bothwomen and men. Moreover, the responsiveness of serum insulin to theglucose tolerance test was decreased by 13% with no change in theglucose response, thus leading to a 34% improvement in the insulinsensitivity index following DHEA administration. An improvement in DHEAaction has also been found in middle-aged men suffering fromhypercholesterolemia (Kawano, Yasue et al. 2003).

In a previous study performed by the same group, DHEA administration for6 months decreased total body fat mass by 1.4 kg while fat-free mass wasincreased by 0.9 kg (Villareal, Holloszy et al. 2000).

Of 25 randomized small size clinical trials enrolling 1353 elderly menwith a men follow-up of 36 weeks, DHEA was associated with a decrease offat mass which was strictly associated with its conversion into itsbiologically active androgen metabolites (Corona, Rastrelli et al.2013). No significant effect was seen on lipid and glycemic metabolism,bone, sexual function and quality of life.

DHEA and Sexual Function

Community-based studies suggest self-reported sexual dysfunctions inwomen which range from 8% to 50%. In fact, low libido and sexualdysfunction increases with age in women from the third decade (Laumann,Paik et al. 1999) as well as after ovariectomy (Nathorst-Boos and vonSchoultz 1992). While phychosocial and health factors are involved inlow arousal and sexual desire (Dennerstein, Dudley et al. 1997) it isbelieved that low androgens play an independent role (Bachmann, Bancroftet al. 2002; Miller, Rosner et al. 2004).

Androgens are known to play a role in women's arousability, pleasure aswell as intensity and ease of orgasm. Androgens are also involved in theneurovascular smooth muscle response of swelling and increasedlubrication (Basson 2004).

In addition, the detailed benefits of androgens added to ERT or HRT havebeen described on general well-being, energy, mood, and general qualityof life (Sherwin and Gelfand 1985; Sherwin 1988). Improvements in themajor psychologic and psychomatic symptoms, namely irritability,nervousness, memory, and insomnia have been observed following additionof androgens to estrogen replacement therapy (ERT) (Notelovitz, Watts etal. 1991).

Loss of libido and/or sexual satisfaction are common in earlypostmenopause. The addition of androgens to hormone replacement therapy(HRT) is known to have beneficial effects on these problems. (Shifren,Braunstein et al. 2000) have found that transdermal testosteroneadministered by patch improved sexual frequency, pleasure and mood insurgically menopausal women. The effect was seen at a daily 300 μg doseof testosterone, a dose that led to serum testosterone levels in theupper limit of normal. Testosterone treatment has also been studied innon androgen-deficient women complaining of decreased libido (Goldstat,Briganti et al. 2003). Such treatment with testosterone improved libido,sexual function as well as quality of life compared to placebo.Similarly, in menopausal women with normal levels of androgens, theaddition of methyltestosterone to estrogen increased sexual desire andfrequency as compared to estrogen alone (Lobo, Rosen et al. 2003). Amongwomen with dysfunction of sexual interest, desire, androgen therapy hasbeen suggested for those having free serum testosterone levels withinthe lower quantile of the reference range (Bachmann, Bancroft et al.2002). In fact, there is increased use of testosterone to treathypoactive sexual desire disorder (HSDD) (Sherwin and Gelfand 1987;Davis, McCloud et al. 1995; Shifren, Braunstein et al. 2000; Goldstat,Briganti et al. 2003). These randomized clinical trials demonstrate thattestosterone is effective in women with HSDD.

A clear example of nature of androgen deficiency of adrenal origin isprovided by cases of adrenal insufficiency. (Arlt, Callies et al. 1999)have studied the effect of DHEA, 50 mg daily and placebo for 4 months ina population of women suffering from adrenal insufficiency. Treatmentwith DHEA raised serum testosterone in the low normal range. Suchtreatment increased the frequency of sexual thoughts, interest andsatisfaction. Well-being, depression and anxiety were also improved. Ina study where DHEA was administered at a high 300 mg daily dose, agreater subjective mental (p<0.016) and physical (p<0.030) was observedin response to an erotic video (Hackbert and Heiman 2002).

Since it is now understood that serum testosterone does not reflect thetotal androgen pool (Labrie, Bélanger et al. 2006), it is not surprisingthat serum testosterone needs to be increased to supraphysiologicallevels to improve sexual function since the serum levels represent onlya fraction of total androgens, which are up to 50% made intracellularlyand not reflected by circulating testosterone levels.

Since androgens appear so crucial for sexual dysfunction in women andpractically 100% of androgens in women originate from DHEA (Labrie2010a; Labrie, Martel et al. 2011) and women benefit from DHEAadministration (Labrie, Archer et al. 2009a), it is reasonable tobelieve that DHEA administration in men having symptoms of loss oflibido and sexual dysfunction (or other symptoms of androgen deficiency)in the presence or absence of low serum testosterone will similarly havebeneficial effects from DHEA administration.

DHEA and Cardiovascular Disease

There is convincing evidence that androgens have beneficial effects oncardiovascular disease (CVD) in men (Alexandersen, Haarbo et al. 1996;Anker, Chua et al. 1997) (Beer, Jakubowicz et al. 1996; Anker, Clark etal. 1997; Hak, Witteman et al. 2002). This is in agreement with theobservation that high serum DHEA is associated with decreased deaths andCVD (Alexandersen, Haarbo et al. 1996).

Clinical trials suggest that testosterone replacement therapy in men mayhelp testosterone deficient men with angina (English, Steeds et al.2000; Malkin, Pugh et al. 2004), congestive cardiac failure (Pugh, Joneset al. 2004; Malkin, Pugh et al. 2006) and type 2 diabetes (Kapoor,Malkin et al. 2005; Kapoor, Goodwin et al. 2006). Moreover, in thehuman, data indicate that DHEA inhibits atherosclerosis (Eich, Nestleret al. 1993; Kurzman, Panciera et al. 1998; Hayashi, Esaki et al. 2000;Komesaroff 2008), reduces cardiovascular risk markers (Mortola and Yen1990; Beer, Jakubowicz et al. 1996) and improves endothelial function(Kawano, Yasue et al. 2003; Williams, Dawood et al. 2004). A protectiverole of DHEA against atherosclerosis has also been observed in primates(Christopher-Hennings, Kurzman et al. 1995) and is particularly wellknown in rabbits (Gordon, Bush et al. 1988; Eich, Nestler et al. 1993).

Apart from the TOM trial, metaanalysis of a series of trials did notshow adverse cardiovascular outcome (Calof, Singh et al. 2005; Haddad,Kennedy et al. 2007; Fernandez-Balsells, Murad et al. 2010). Shores etal, 2012 observed a 39% decrease in mortality risk in patients treatedwith testosterone and a 20% lower incidence of heart disease (Shores,Smith et al. 2012).

In the Testosterone in Older Men with Mobility Limitation (TOM) trial,the men who experienced cardiovascular events had greater increases inserum free testosterone level than those who did not (Basaria, Davda etal. 2013).

Low serum DHEA-S has been found to be positively associated with theincidence of cardiovascular events (Mitchell, Sprecher et al. 1994), theextent (Herrington, Gordon et al. 1990) as well as the incidence(Herrington, Nanjee et al. 1996), of angiographic coronary stenosis,thus suggesting a protective role of DHEA-S on CVD. Moreover, low serumtestosterone has been associated with an increased risk of coronaryartery disease in men (Turhan, Tulunay et al. 2007) while low DHEAlevels have been reported to predispose to earlier death from CVD(Barrett-Connor, Khaw et al. 1986; Tivesten, Vandenput et al. 2009;Ohlsson, Labrie et al. 2010).

DHEA and the Brain

In addition to the traditional symptoms of menopause (Raven and Hinson2007), the DHEA decline with age has been linked to loss of memory andcognitive function (Flood and Roberts 1988; Grimley Evans, Malouf et al.2006).

A role of DHEA has been proposed in the etiology and treatment ofneuronal damage induced by Alzheimer's disease (Simpkins, Green et al.1997; Weill-Engerer, David et al. 2002; Yau, Rasmuson et al. 2003). Thehippocampus is a brain region involved in learning, cognition andmemory. This brain area shows pronounced changes during aging and inAlzheimer's disease (Beck and Handa 2004). Estrogens and DHEA which canform estrogens locally in the brain have been shown to enhance memoryand learning functions (McEwen, Gould et al. 1995; Foy 2001; Vallee,Mayo et al. 2001). Studies have shown that DHEA-S can influence brainfunction and positively affect memory mood and energy and indirectlyphysical activity (Wolkowitz, Reus et al. 1999; Hunt, Gurnell et al.2000; Huppert and Van Niekerk 2001).

the human, tests of long-term memory have been improved by DHEAadministration (Barrett-Connor and Edelstein 1994). In addition, theoral administration of 25 mg DHEA per day for 12 months in aging maleswith partial androgen deficiency improved mood and fatigue in additionto joint pain (Genazzani, Inqlese et al. 2004).

A role of androgens has been proposed on depression, memory loss, lossof cognition and brain cell activity (Azad, Pitale et al. 2003; Hajszan,MacLusky et al. 2007; Almeida, Yeap et al. 2008). Estrogens which canalso be synthesized in brain from DHEA have been shown to have abeneficial role in Alzheimer's disease, memory loss and loss ofcognition (Rocca, Bower et al. 2007). Three metaanalyses have shown a 20to 40% decreased risk of Alzheimer's disease in women who used estrogenafter menopause (Yaffe 1998; Hogervorst, Williams et al. 2000; LeBlanc,Janowsky et al. 2001). Estrogen reduces beta-amyloid deposition in thebrain whereas progesterone has the opposite effect (Xu, Gouras et al.1998; Huanq, Guan et al. 2004). There is now solid evidence fromclinical studies that there is a critical age window for the beneficialeffects of estrogens on neuroprotection (Rocca, Bower et al. 2007),cardiovascular disease (Manson, Bassuk et al. 2006) and overallmortality (Rocca, Grossardt et al. 2006).

An association between lack of estrogen and cognitive impairment ordementia is supported by laboratory data. Among them estrogen improvessynapse formation on dendritic spines in the hippocampi ofoophorectomized rats (McEwen and Alves 1999; Monk and Brodaty 2000).Moreover, estrogen improves cerebral blood flow and glucose metabolismand it may act as an antioxidant (Gibbs and Aggarwal 1998; McEwen andAlves 1999; Monk and Brodaty 2000). Estrogen has also been found toprevent B-Amyloid 1-42 from inducing a rise in intracellular calcium andfrom causing mitochondrial damage (Chen, Nilsen et al. 2006; Morrison,Brinton et al. 2006).

More and more evidence suggests a role of sex steroids, namely estradioland testosterone in neuroprotection on the brain (Pike, Carroll et al.2009). Data from cell culture and animal studies support testosterone asneuroprotective (Holland, Bandelow et al. 2011) and same data suggests abeneficial effect in older men on cognition (Tan and Pu 2003). In arecent preclinical study, testosterone reduced neuronal and vascularaging in hippocampal cells while (Ota, Akishita et al. 2012) decreasingcognitive decline.

Lower serum testosterone levels were found in old men with Alzheimer'sdisease compared to controls (Hogervorst, Bandelow et al. 2004).

Low DHEA in Longevity

Low DHEA-S has been associated to low longevity (Kushnir, Blamires etal. 2010; Labrie 2010b; Arauio and Wittert 2011; Traish, Kanq et al.2011; Maggi, Buvat et al. 2013).

Other Potential Benefits of DHEA

The 70 to 95% reduction in the formation of DHEA and DHEA-S by theadrenals during aging results in a dramatic reduction in the formationof androgens and estrogens in peripheral target tissues, which couldwell be involved in the pathogenesis of age-related diseases such asinsulin resistance (Coleman, Leiter et al. 1982; Schriock, Buffington etal. 1988) and obesity (Nestler, Barlascini et al. 1988; MacEwen andKurzman 1991; Tchernof, Desprès et al. 1995). DHEA has been found toexert antioncogenic activity in a series of animal models (Schwartz,Pashko et al. 1986; Gordon, Shantz et al. 1987; Li Yan et al. 1993).DHEA has also been shown to have immuno modulatory effects in vitro(Suzuki, Suzuki et al. 1991) and in vivo in fungal and viral diseases(Rasmussen, Arrowood et al. 1992), including HIV (Henderson, Yang et al.1992). On the other hand, a stimulatory effect of DHEA on the immunesystem has been described in postmenopausal women (Casson, Andersen etal. 1993).

DHEA and Lipids

Following administration of various doses of DHEA for variable periodsof time, small but significant decreases in total and high-densitylipoprotein (HDL) cholesterol have been reported (Nestler, Barlascini etal. 1988; Mortola and Yen 1990; Arlt, Callies et al. 1999; Barnhart,Freeman et al. 1999; Petri, Lahita et al. 2002; Petri, Mease et al.2004) while, in other studies, low-density lipoprotein (LDL) cholesterolwas also decreased in addition to total and HDL cholesterol(Gebre-Medhin, Husebye et al. 2000; Dhatariya, Bigelow et al. 2005). Asmall decrease in serum HDL cholesterol has been reported in previousstudies with DHEA administered at the daily dose of 50 mg (Arlt, Callieset al. 1999; Barnhart, Freeman et al. 1999; Hunt, Gurnell et al. 2000),4-6 g 10% DHEA cream (Labrie, Bélanger et al. 1997a), 1600 mg (Mortolaand Yen 1990) or 25 mg (Casson, Santoro et al. 1998) while, in otherstudies, no significant effect was seen at 50 mg/day (Morales, Nolan etal. 1994; Barnhart, Freeman et al. 1999; Villareal, Holloszy et al.2000) or 25 mg/day (Kawano, Yasue et al. 2003; Lovas, Gebre-Medhin etal. 2003).

DHEA, contrary to estrogens, does not increase triglycerides (Diamond,Cusan et al. 1996). In fact, a decrease in triglycerides is often seenwith DHEA (Lasco, Frisina et al. 2001; Chang, Lan et al. 2002;Dhatariya, Bigelow et al. 2005). Increased HDL and decreased LDLcholesterol have also been reported (Lasco, Frisina et al. 2001) while adecrease in total cholesterol only has been reported (Libe, Barbetta etal. 2004; Williams, Dawood et al. 2004). DHEA administration inpostmenopausal women has also been reported to decrease serumApolipoprotein A and increase HDL cholesterol (Casson, Santoro et al.1998; Morales, Haubrich et al. 1998). DHEA has been found to decreaseserum Lp(A) (Barnhart, Freeman et al. 1999), an effect which should bebeneficial for CVD (Lobo 1991).

The decrease in triglycerides and HDL cholesterol levels under theinfluence of androgens has been reported to result from increasedhepatic lipase activity which results in increased clearance of HDL(Haffner, Kushwaha et al. 1983; Hazzard, Haffner et al. 1984; Kantor,Bianchini et al. 1985). The increased reverse cholesterol transport(removal of cholesterol from peripheral tissues via increased HDLclearance) seems responsible for the decreased HDL and triglyceridelevels rather than decreased HDL production (Wu and von Eckardstein2003). The relatively small (when present) inhibitory effect of DHEA ontotal cholesterol, HDL cholesterol and sometimes LDL cholesterol couldalso involve the effects of DHEA-derived androgens on hepatic lipaseactivity, thus impairing hepatic cholesterol formation (Tan, Shiu et al.1998).

The consensus is that DHEA has only small and no clinically significanteffects on lipids (Arlt, Justl et al. 1998; Morales, Haubrich et al.1998; Gebre-Medhin, Husebye et al. 2000; Lasco, Frisina et al. 2001;Poretsky, Brillon et al. 2006; Gurnell, Hunt et al. 2008; Lovas andHusebye 2008). Our preclinical studies, however, shown inhibitory effecton serum triglycerides and no effect on cholesterol (FIG. 10) whileEM-800 (a prodrug of acolbifene, free salt) decreases both serumtriglycerides (FIG. 10) and cholesterol (FIGS. 10 and 11).

Benefits of DHEA: Combination of Estrogen-Like and Androgenic Effects

The present invention is based upon the recent progress achieved in ourunderstanding of sex steroid physiology in men and women and therecognition that women, at menopause, are not only deprived fromestrogen due to the arrest of estrogen secretion by the ovaries, buthave already been submitted for a few years to a decreasing exposure toandrogens. In fact, normal women produce an amount of androgensequivalent to approximately 50% of the androgens secreted in men(Labrie, Bélanger et al. 1997a). The pool of androgens in men and womendecreases progressively from the age of 30 years in parallel with thedecrease in the serum concentration of DHEA and DHEA-S (Labrie, Bélangeret al. 1997b). The addition of a SERM like acolbifene is to increase theserum levels of testosterone (FIG. 12) as well as the positive effect onbone loss protection as well as on other benefits of SERMadministration. In FIG. 12, a schematic representation of the effects ofDHEA and acolbifene is presented by blocking the negative feedbackeffect of estrogens on GnRH/LH secretion, as further illustrated inFIGS. 13 and 14 obtained in the cynomolgus male monkey, increased serumtestosterone levels are observed.

That acolbifene increased LH secretion in the human is indicated by theincrease in serum E₂ levels from 222 to 2030 μg/mL at 6 months of dailyoral administration of 20 mg acolbifene in a premenopausal women withadvanced breast cancer.

Beneficial Effects of Acolbifene

It can be seen in FIG. 15 that the approximately 100% stimulatory effectof tamoxifen on tumor growth was completely blocked by simultaneoustreatment with EM-652.HCl (acolbifene) EM-652.HCl in accordance with itspure antiestrogenic activity did not exert any stimulatory effect on thegrowth of the human breast cancer ZR-75-1 xenografts in nude mice.

We have also noted a correlation between the beneficial effect of SERMshave on serum cholesterol and beneficial estrogenic or estrogen-likeeffects on bone. SERMs have also a beneficial effect on hypertension,insulin resistance, diabetes, and obesity (especially abdominalobesity). Without intending to be bound by theory, it is believed thatSERMs, many of which preferably have two aromatic rings linked by one totwo carbon atoms, are expected to interact with the estrogen receptor byvirtue of the foregoing portion of the molecule that is best recognizedby the receptor. Preferred SERMs have side chains which may selectivelycause antagonistic properties in breast and usually uterine tissueswithout having significant antagonistic properties in other tissues.Thus, the SERMs may desirably functions as antiestrogens in the breastwhile surprisingly and desirably functioning as estrogens (or providingestrogen-like activity) in bone and in the blood (where concentrationsof lipid and cholesterol are favorably affected). The favorable effecton cholesterol and lipids translates to a favorable effect againstatherosclerosis which is known to be adversely, affected by improperlevels of cholesterol and lipids (FIGS. 10 and 11).

Cardiovascular symptoms, Alzheimer's disease, loss of cognitivefunctions and insomnia involve certainly estrogen receptors situated inthe nervous central system. Probably, decreased levels of estrogens (orandrogens) in the brain, can explain at least in part, these conditions.Exogenous estrogens and particularly those (i.e. estradiol) formed bythe administration of sex steroid precursors can pass through the brainbarrier and bind to the estrogen receptor to restore the normalestrogenic action. On the other hand, SERMs of the invention, and moreparticularly those of acolbifene family, cannot pass through the brainbarrier as shown in example 8. Thus, they cannot antagonise the positiveeffect of estrogens in brain but they antagonise the negative effects ofestrogens in the breast, rending this combination (SERM+sex steroidprecursor) particularly attractive for the treatment or reduction of therisk of acquiring the above-mentioned conditions.

As mentioned earlier, a role for androgens has also been suggested forall these symptoms. In fact, DHEA can provide both estrogens andandrogens in the brain according to physiological needs.

Overall Additive Benefits of Combining a Sex Steroid Precursor and aSERM or an Antiestrogen

No adverse effect of EM-652 (acolbifene) has been seen on any parameterwhile it should exert marked beneficial effects for the prevention andtreatment of gynecomastia, breast cancer and osteoporosis.

Preferred SERMs or antiestrogens discussed herein relate: (1) to alldiseases stated to be susceptible to the invention; (2) to boththerapeutic and prophylactic applications; and (3) to preferredpharmaceutical compositions and kits.

A patient in need of treatment or of reducing the risk of onset of agiven disease is one who has either been diagnosed with such disease orone who is susceptible of acquiring such disease.

Except where otherwise stated, the preferred dosage of the activecompounds (concentrations and modes of administration) of the inventionis identical for both therapeutic and prophylactic purposes. The dosagefor each active component discussed herein is the same regardless of thedisease being treated (or of the disease whose likelihood of onset isbeing reduced).

Except when otherwise noted or where apparent from context, dosagesherein refer to weight of active compounds unaffected by pharmaceuticalexcipients, diluents, carriers or other ingredients, although suchadditional ingredients are desirably included, as shown in the examplesherein. Any dosage form (capsule, pill, tablet, injection or the like)commonly used in the pharmaceutical industry is appropriate for useherein, and the terms “excipient”, “diluent”, or “carrier” include suchnonactive ingredients as are typically included, together with activeingredients in such dosage forms in the industry. For example, typicalcapsules, pills, enteric coatings, solid or liquid diluents orexcipients, flavorants, preservatives, or the like may be included.

All of the active ingredients used in any of the therapies discussedherein may be formulated in pharmaceutical compositions which alsoinclude one or more of the other active ingredients. Alternatively, theymay each be administered separately but sufficiently simultaneous intime so that a patient eventually has elevated blood levels or otherwiseenjoys the benefits of each of the active ingredients (or strategies)simultaneously. In some preferred embodiments of the invention, forexample, one or more active ingredients are to be formulated in a singlepharmaceutical composition. In other embodiments of the invention, a kitis provided which includes at least two separate containers wherein thecontents of at least one container differs, in whole or in part, fromthe contents of at least one other container with respect to activeingredients contained therein.

Combination therapies discussed herein also include use of one activeingredient (of the combination) in the manufacture of a medicament forthe treatment (or risk reduction) of the disease in question where thetreatment or prevention further includes another active ingredient ofthe combination in accordance with the invention. For example in oneembodiment, the invention provides the use of a SERM in the preparationof a medicament for use, in combination with a sex steroid precursor invivo, in the treatment of any of the diseases for which the presentcombination therapy is believed effective.

The limitations of bone mineral density (BMD) measurements are wellknown. As an example, BMD measurements showed no change in rats treatedwith the steroidal antiestrogen ICI 182780 (Wakeling 1993) whileinhibitory changes were seen by histomorphometry (Gallagher, Chambers etal. 1993). Similar differences were reported with tamoxifen (Jordan,Phelps et al. 1987; Sibonga, Evans et al. 1996).

It should be indicated that reduced bone mineral density is not the onlyabnormality associated with reduced bone strength. It is thus importantto analyze the changes in biochemical parameters of bone metabolisminduced by various compounds and treatments in order to gain a betterknowledge of their action (Table 2).

It is particularly important to indicate that the combination of DHEAand acolbifene exerted unexpected beneficial effects on importantbiochemical parameters of bone metabolism. In fact, DHEA alone did notaffect the urinary hydroxyproline/creatinine ratio, a marker of boneresorption. Moreover, no effect of DHEA could be detected on dailyurinary calcium or phosphorus excretion (Luo, Sourla et al. 1997).EM-800 (prodrug of acolbifene free salt) decreased the urinaryhydroxyproline/creatinine ratio by 48% while, similarly to DHEA, noeffect of EM 800 (prodrug of acolbifene, free salt) was seen on urinarycalcium or phosphorus excretion. EM-800, moreover, had no effect onserum alkaline phosphatase activity, a marker of bone formation whileDHEA increased the value of the parameter by about 75% (Luo, Sourla etal. 1997).

One of the unexpected effects of the combination of DHEA and EM-800relates to the urinary hydroxyproline/creatinine ratio, a marker of boneresorption, which was reduced by 69% when both DHEA and EM-800 werecombined, this value being statistically different (p<0.01) from the 48%inhibition achieved by EM-800 alone while DHEA alone did not show anyeffect. Thus, the addition of DHEA to EM-800 increases by 50% theinhibitory effect of EM-800 on bone reabsorption. Most importantly,another unexpected effect of the addition of DHEA to EM-800 (prodrug ofacolbifene, free salt) was the approximately 84% decrease in urinarycalcium (from 23.17±1.55 to 3.71±0.75 μmol/24 h/100 g (p<0.01) and the55% decrease in urinary phosphorus (from 132.7±6.08 to 59.06±4.76μmol/24 h/100 g (p<0.01) respectively (Luo, Sourla et al. 1997).

Importantly, the combination of acolbifene and DHEA in ovariectomizedrats treated for 12 months had beneficial effects on bone morphometry.Trabecular bone volume is particularly important for bone strength andto prevent bone fractures (FIG. 7). Thus, in the above-mentioned study,trabecular bone volume of the tibia increased from 4.1±0.7% inovariectomized rats to 11.9±0.6% (p<0.01) with DHEA alone while theaddition of EM-800 to DHEA further increased trabecular bone volume to14.7±1.4%, a value similar to that found in intact controls (FIG. 7).

From a value of 0.57±0.08 per mm in ovariectomized rats, treatment withDHEA resulted in a 137% increase in trabecular bone number compared toovariectomized controls (FIG. 8). The stimulatory effect of DHEA thusreached 1.27±0.1 per mm while simultaneous treatment with EM-800 andDHEA resulted in an additional 28% increase in trabecular bone number(p<0.01) compared to that achieved by DHEA alone (FIG. 8). Similarly,the addition of EM-800 to DHEA treatment, resulted in an additional 15%(p<0.05) decrease in trabecular bone separation, compared to thatachieved with DHEA alone, thus leading to values not different fromthose seen in intact controls.

As complement to the numerical data presented in FIGS. 7 and 8, FIG. 9illustrates the increase in trabecular bone volume in the proximal tibiametaphysis induced by DHEA in ovariectomized treated animals (C)compared to ovariectomized controls (B), as well as the partialinhibition of the stimulatory effect of DHEA after the addition ofFlutamide to DHEA treatment (D). On the other hand, administration ofDHEA in combination with EM-800 resulted in a complete prevention of theovariectomy-induced osteopenia (E), the trabecular bone volume beingcomparable to that seen in intact controls (A).

TABLE 2 URINE SERUM CALCIUM PHOSPHORUS HP/Cr tALP GROUP (μmol/24 h/100g) (μmol/24 h/100 g) (μmol/mmol) (IU/L) CONTROL 23.17 ± 1.55  132.72 ±6.08  13.04 ± 2.19  114.25 ± 14.04  DHEA (10 mg) 25.87 ± 3.54  151.41 ±14.57  14.02 ± 1.59  198.38 ± 30.76* EM-800 (75 μg) 17.44 ± 4.5  102.03± 25.13    6.81 ± 0.84** 114.11 ± 11.26  DHEA + EM-800  3.71 ± 0.75** 59.06 ± 4.76**   4.06 ± 0.28**  204.38 ± 14.20**

TABLE 3 Effect of 12-month treatment with dehydroepiandrosterone (DHEA)administered alone or in combination with Flutamide (FLU) or EM-800 onbone markers and serum lipids. Alkaline OH- phospha- proline/ Trigly-tase creatinine Cholesterol cerides Group IU/L μmol/mmol mmol/L mmol/LIntact Control  30 ± 3** 15.4 ± 1.3  2.28 ± 0.12 1.4 ± 0.2 OVX Control51 ± 4  11.7± 1.2  2.29 ± 0.16 1.1 ± 0.1 OVX + DHEA  201 ± 25**  7.3 ±1.0*  1.78 ± 0.16* 0.8 ± 0.1 OVX + DHEA +  103 ± 10** 14.5 ± 1.2  2.27 ±0.15 0.8 ± 0.1 FLU OVX + DHEA +  202 ± 17**   6.4 ± 1.0**  0.63 ± 0.09**1.0 ± 0.2 EM-800 *p < 0.05; **p < 0.01 versus OVX Control

The importance of the androgenic component of the stimulatory effect ofDHEA on bone histomorphometry is also supported by the effect of DHEA onmarkers of bone formation and resorption. The concentration of serumalkaline phosphatase, a marker of bone formation (Lauffenburger, Olah etal. 1977; Meunier, Salson et al. 1987) was increased from 51±4 IU/L inOVX controls to 201±25 IU/L in DHEA-treated animals, suggesting astimulatory effect of DHEA on bone formation (Table 3). FLU reversed by65% the stimulatory effect of DHEA on this parameter while EM-800 had nosignificant effect. On the other hand, since hydroxyproline releasedduring collagen degradation is not reutilized in collagen synthesis, itis a useful marker of collagen metabolism or osteoclastic boneresorption. In the present study, the urinary hydroxyproline/creatinineratio decreased from 11.7±1.2 μmol/mmol in OVX controls to 7.3±1.0μmol/mmol (p<0.05) in DHEA-treated rats (Table 3). The administration ofFLU completely prevented the inhibitory effect of DHEA on this parameterwhile EM-800 had no statistically significant influence on the effect ofDHEA.

Moreover, serum cholesterol was reduced by 22% from 2.29±0.16 to1.78±0.16 mmol/l (p<0.05) by DHEA treatment, an effect neutralized byconcomitant treatment with the pure antiandrogen FLU. The addition ofthe pure antiestrogen EM-800, on the other hand, decreased total serumcholesterol further to 0.63±0.09 mmol/l (p<0.01), thus reaching a 65%inhibitory effect. No statistically significant change was observed inserum triglyceride levels with any of the treatments used (Table 3).

It is also of interest to note that the potent inhibitory effect ofEM-800 (prodrug of acolbifene, free salt) on serum cholesterol is notprevented by simultaneous treatment with DHEA (Luo, Sourla et al. 1997).

Cancellous bone strength and subsequent resistance to fracture do notonly depend upon the total amount of cancellous bone but also on thetrabecular microstructure, as determined by the number, size, anddistribution of the trabeculae. The loss of ovarian function inpostmenopausal women is accompanied by a significant decrease in totaltrabecular bone volume (Melsen, Melsen et al. 1978; Kleerekoper,Villanueva et al. 1985), mainly related to a decrease in the number and,to a lesser degree, in the width of trabeculae (Weinstein and Hutson1987).

In order to facilitate the combination therapy aspect of the invention,for any indication discussed herein, the invention contemplatespharmaceutical compositions which include the SERM and the sex steroidprecursor in a single composition for simultaneous administration. Thecomposition may be suitable for administration in any traditional mannerincluding but not limited to oral administration, subcutaneousinjection, intramuscular injection or percutaneous administration. Inother embodiments, a kit is provided wherein the kit includes one ormore SERM and sex steroid precursor in separate or in one container. Thekit may include appropriate materials for oral administration, e.g.tablets, capsules, syrups and the like and for transdermaladministration, e.g., ointments, lotions, gels, creams, sustainedrelease patches and the like.

Applicants believe that administration of SERMs or antiestrogens and sexsteroid precursors has utility in the treatment and/or reduction of theincidence of any of the symptoms mentioned above. The active ingredientsof the invention (whether SERM, antiestrogen or precursor or otherwise)may be formulated and administered in a variety of ways. Whenadministered together in accordance with the invention, the activeingredients may be administered simultaneously or separately.

Active ingredient for transdermal or transmucosal is preferably from0.01% to 5%, DHEA or 5-diol.

That the SERM can be administered percutaneously is indicated by thecomparable efficacy of acolbifene analogs to antagonize the stimulatoryeffect of estradiol on uterine weight whether acolbifene analogs areadministered orally or percutaneously in mice (FIG. 16).

When formulated as an ointment, lotion, gel, cream, or suppository orthe like, the active compound is admixed with a suitable carrier whichis compatible with human skin or mucosa and which enhances transdermalor transmucosal penetration of the compound through the skin or mucosa.Suitable carriers are known in the art and include but are not limitedto Klucel HF and Glaxal base. Some are commercially available, e.g.,Glaxal base available from Glaxal Canada Limited Company. Other suitablevehicles can be found in Koller and Buri, S.T.P. Pharma (Kroller andBuri 1987). The carrier is preferably one in which the activeingredient(s) is (are) soluble at ambient temperature at theconcentration of active ingredient that is used. The carrier should havesufficient viscosity to maintain the inhibitor on a localized area ofskin or mucosa to which the composition has been applied, withoutrunning or evaporating for a time period sufficient to permitsubstantial penetration of the precursor through the localized area ofskin or mucosa and into the bloodstream where it will cause a desirableclinical effect. The carrier is typically a mixture of severalcomponents, e.g. pharmaceutically acceptable solvents and a thickeningagent. A mixture of organic and inorganic solvents can aid hydrophylicand lipophylic solubility, e.g. water and an alcohol such as ethanol.

When formulated as an ovule or a rectal suppository or the like, theactive compound is admixed with a suitable carrier which is compatiblewith human rectal mucosa. Preferred carriers are hard fats (mixture ofglycerides of saturated fatty acids), particularly Witepsol, andspecially Witepsol H-15 base (available from Medisca, Montreal, Canada).Any other lipophilic base such as Fattibase, Wecobee, cocoa butter,theobroma oil or other combinations of Witepsol bases could used.

Preferred sex steroid precursors are dehydroepiandrosterone (DHEA)(available, for example, from Proquina, Orizaba, Veracruz, Mexico).

The carrier may also include various additives commonly used inointments, lotions and suppositories and well known in the cosmetic andmedical arts. For example, fragrances, antioxidants, perfumes, gellingagents, thickening agents such as carboxymethylcellulose, surfactants,stabilizers, emollients, coloring agents and other similar agents may bepresent.

Treatment in accordance with the invention is suitable for indefinitecontinuation. The SERM, or antiestrogenic compound and the sex steroidprecursor can also be administered, by the oral route, and may beformulated with conventional pharmaceutical excipients, e.g. spray driedlactose, microcrystalline cellulose, and magnesium stearate into tabletsor capsules for oral administration.

The active substances can be worked into tablets or dragee cores bybeing mixed with solid, pulverulent carrier substances, such as sodiumcitrate, calcium carbonate or dicalcium phosphate, and binders such aspolyvinyl pyrrolidone, gelatin or cellulose derivatives, possibly byadding also lubricants such as magnesium stearate, sodium laurylsulfate, “Carbowax” or polyethylene glycol. Of course, taste-improvingsubstances can be added in the case of oral administration forms.

As further forms, one can use plug capsules, e.g. of hard gelatin, aswell as closed soft-gelatin capsules comprising a softener orplasticizer, e.g. glycerin. The plug capsules contain the activesubstance preferably in the form of granulate, e.g. in mixture withfillers, such as lactose, saccharose, mannitol, starches, such as potatostarch or amylopectin, cellulose derivatives or highly dispersed silicicacids. In solf-gelatin capsules, the active substance is preferablydissolved or suspended in suitable liquids, such as vegetable oils orliquid polyethylene glycols.

The lotion, ointment, gel or cream should be thoroughly rubbed into theskin so that no excess is plainly visible, and the skin should not bewashed in that region until most of the transdermal penetration hasoccurred preferably at least 4 hours and, more preferably, at least 6hours.

A transdermal patch may be used to deliver precursor in accordance withknown techniques. It is typically applied for a much longer period,e.g., 1 to 4 days, but typically contacts active ingredient to a smallersurface area, allowing a slow and constant delivery of activeingredient.

A number of transdermal drug delivery systems that have been developed,and are in use, are suitable for delivering the active ingredient of thepresent invention. The rate of release is typically controlled by amatrix diffusion, or by passage of the active ingredient through acontrolling membrane.

Mechanical aspects of transdermal devices are well known in the rat, andare explained, for example, in U.S. Pat. Nos. 5,162,037, 5,154,922,5,135,480, 4,666,441, 4,624,665, 3,742,951, 3,797,444, 4,568,343,5,064,654, 5,071,644, 5,071,657, the disclosures of which areincorporated herein by reference. Additional background is provided byEuropean Patent 0279982 and British Patent Application 2185187.

The device may be any of the general types known in the art includingadhesive matrix and reservoir-type transdermal delivery devices. Thedevice may include drug-containing matrixes incorporating fibers whichabsorb the active ingredient and/or carrier. In a reservoir-type device,the reservoir may be defined by a polymer membrane impermeable to thecarrier and to the active ingredient.

In a transdermal device, the device itself maintains active ingredientin contact with the desired localized skin surface. In such a device,the viscosity of the carrier for active ingredient is of less concernthan with a cream or gel. A solvent system for a transdermal device mayinclude, for example, oleic acid, linear alcohol lactate and dipropyleneglycol, or other solvent systems known in the art. The active ingredientmay be dissolved or suspended in the carrier.

For attachment to the skin, a transdermal patch may be mounted on asurgical adhesive tape having a hole punched in the middle. The adhesiveis preferably covered by a release liner to protect it prior to use.Typical material suitable for release includes polyethylene andpolyethylene-coated paper, and preferably silicone-coated for ease ofremoval. For applying the device, the release liner is simply peeledaway and the adhesive attached to the patient's skin. In U.S. Pat. No.5,135,480, the disclosure of which is incorporated by reference,describe an alternative device having a non-adhesive means for securingthe device to the skin.

It is necessary only that SERM, antiestrogen and sex steroid precursorbe administered in a manner and at a dosage sufficient to allow bloodserum concentration of each to obtain desired levels. In accordance withthe combination therapy of the invention, concentration of the SERM ismaintained within desired parameters at the same time that sex steroidprecursor concentration is maintained within desired parameters

One preferred sex steroid precursor is DHEA, although DHEA-S and analogsdiscussed below are also especially effective for the reasons statedbelow.

A selective estrogen receptor modulator of the invention has a molecularformula with the following features: a) two aromatic rings spaced by 1to 2 intervening carbon atoms, both aromatic rings being eitherunsubstituted or substituted by a hydroxyl group or a group converted invivo to hydroxyl; and b) a side chain possessing an aromatic ring and atertiary amine function or salt thereof.

One preferred SERM of the invention is acolbifene:

Acolbifene (also called EM-652.HCl; EM-1538) is the hydrochloride saltof the potent antiestrogen EM-652. It is disclosed in U.S. Pat. No.6,710,059 B1. Another preferred SERM is lasofoxifene (Oporia;CP-336,156;(−)-cis-(5R,6S)-6-phenyl-5-[4-(2-pyrrolidin-1-ylethoxy)phenyl]-5,6,7,8-tetrahydronaphthalen-2-ol,D-(−)-tartrate salt) (available from Pfizer Inc., USA).

Another preferred SERM is bazedoxifene (TSE 424; WAY-TSE 424; WAY140424;1-[[4-[2-(hexahydro-1H-azepin-1-yl)ethoxy]phenyl]methyl]-2-(4-hydroxyphenyl)-3-methyl-1H-indol-5-ol, acetate) developed by Wyeth Ayers (USA)and disclosed in JP10036347 (American home products corporation) andapproved in USA for the prevention of postmenopausal osteoporosis andnon-steroidal estrogen derivatives described in WO 97/32837. Otherpreferred SERMs of the invention include tamoxifen((Z)-2-[4-(1,2-diphenyl-1-butenyl) phenoxy]-N,N-dimethylethanamine)(available from Zeneca, UK), toremifene((Z)-2-[4-(4-Chloro-1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethanamine)available from Orion, Finland, under the trademark Fareston orSchering-Plough), droloxifene ((E)-3-[1-[4-[2-(Dimethylamino)ethoxy]phenyl]-2-phenyl-1-butenyl]phenol) and, from Eli Lilly and Co.,USA: raloxifene([2-(4-hydroxyphenyl)-6-hydroxybenzo[b]thien-3-yl][4-[2-(1-piperidinyl)ethoxy]phenyl]-methanone hydrochloride), LY 335124, LY 326315, LY 335563(6-hydroxy-3-[4-[2-(1-piperidinyl)ethoxy]phenoxyl]-2-(4-hydroxyphenyl)benzo[b]thiopenehydrochloride) and arzoxifene (LY 353381,6-hydroxy-3-[4-[2-(1-piperidinyl)ethoxy]phenoxyl]-2-(4-methoxyphenyl)benzo[b]thiophenehydrochloride). Other preferred SERMs are idoxifene((E)-1-[2-[4-[1-(4-Iodophenyl)-2-phenyl-1-butenyl]phenoxy]ethyl]pyrrolidine)(SmithKline Beecham, USA), levormeloxifene(3,4-trans-2,2-dimethyl-3-phenyl-4-[4-(2-(pyrrolidin-1-yl)ethoxy)phenyl]-7-methoxychroman)(Novo Nordisk, A/S, Denmark) which is disclosed in Shalmi et al. WO97/25034, WO 97/25035, WO 97/25037, WO 97/25038; and Korsgaard et al. WO97/25036), GW5638 (described by Willson et al., 1997) and indolederivatives (disclosed by Miller et al., EP 0802183A1) Are alsoincluded, Iproxifen (TAT 59;(E)-4-[1-[4-[2-(dimethylamino)ethoxy]phenyl]-2-[4-(1-methylethyl)phenyl]-1-butenyl]phenoldihydrogen phosphate) from Taiho (Japan), ospemifene (FC 1271;((Z)-2-[4-(4-chloro-1,2-diphenyl-1-butenyl)phenoxyl]ethanol) fromavailable from Orion-Farmos Pharmaceutica, Finland, SERM 3471, HMR 3339and HMR 3656 from Sanofi-Aventis (France), pipendoxifene (ERA 923)developed by Wyeth-Ayers, nonsteroidal estrogen derivatives described inWO 97/3283, fispemifene developed by QuatRx (USA) and CC 8490 developedby Celgene in USA.

Any SERM used as required for efficacy, as recommended by themanufacturer, can be used. Appropriate dosages are known in the art. Anyother non steroidal antiestrogen commercially available can be usedaccording to the invention. Any compound having activity similar toSERMs (example: raloxifene can be used).

SERMs administered in accordance with the invention are preferablyadministered in a dosage range between 0.01 to 5 mg/kg of body weightper day (preferably 0.05 to 1.0 mg/kg), with 5 mg per day, especially 10mg per day, in two equally divided doses being preferred for a person ofaverage body weight when orally administered, or in a dosage rangebetween 0.003 to 3.0 mg/kg of body weight per day (preferably 0.015 to0.3 mg/mL), with 1.5 mg per day, especially 3.0 mg per day, in twoequally divided doses being preferred for a person of average bodyweight when parentally administered (i.e. intramuscular, subcutaneous orpercutaneous administration). Preferably the SERMs are administeredtogether with a pharmaceutically acceptable diluent or carrier asdescribed below.

One preferred antiestrogen of the invention is fulvestrant (Faslodex;ICI 182 780;7α-[9-(4,4,5,5,5-pentafluoro-pentylsulphinyl)nonyl]oestra-1,3,5(10)-triene-3,17β-diol)which is intramuscularly administered with the dosage of 250 mg permonth available from AstraZeneca Canada Inc., Mississauga, Ontario,Canada. Other preferred antiestrogen is SH 646 from Schering AG, Germany

With respect to all of the dosages recommended herein, the attendingclinician should monitor individual patient response and adjust dosageaccordingly.

EXAMPLES Example 1 Example of Synthesis of the Preferred Compound of theInvention

Synthesis of acolbifene((S)-(+)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-(2′″-piperidinoethoxy)phenyl)-2H-1-benzopyranhydrochloride, EM-01538, (EM-652.HCl)).

Step A: BF₃.Et₂O, toluene; 100° C.; 1 hour.Step C: 3,4-dihydropyran, p-toluenesulfonic acid monohydrate, ethylacetate; 25° C. under nitrogen, 16 hours, and then crystallization inisopropanol.

Steps D, E, and F:

(1) piperidine, toluene, Dean & Stark apparatus, reflux under nitrogen;(2) 1,8-diazabicyclo[5,4,0]undec-7-ene, DMF, reflux 3 hours;(3) CH₃MgCl, THF, −20 to 0° C. and then room temperature for 24 hours;Steps G, H: (1S)-(+)-10-camphorsulfonic acid, acetone, water, toluene,room temperature, 48 hours.Step HH: 95% ethanol, 70° C., then room temperature 3 days.Step HHR: Recycling of mother liquor and wash of step HH(S)-10-camphorsulfonic acid, reflux; 36 hours, then room temperature for16 hours.

Step I:

(1) DMF aq., Na₂CO₃, ethyl acetate;(2) Ethanol, dilute HCl;

(3) Water. Synthesis of2-tetrahydropyranyloxy-4-hydroxy-2′-(4″-tetrahydropyranyloxy phenyl)acetophenone (4)

A suspension of 2,4-dihydroxy-2′-(4″-hydroxyphenyl)acetophenone 3 (97.6g, 0.4 mole) (available from Chemsyn Science Laboratories, Lenexa,Kans.) in 3,4-dihydropyran (218 mL, 3.39 mole) and ethyl acetate (520mL) was treated with p-toluenesulfonic acid monohydrate (0.03 g, 0.158mmole) at about 25° C. The reaction mixture was stirred under nitrogenwith no external heating for about 16 hours. The mixture was then washedwith a solution of sodium bicarbonate (1 g) and sodium chloride (5 g) inwater (100 mL). The phases were separated and the organic phase waswashed with brine (20 mL). Each wash was back extracted with 50 mL ethylacetate. All the organic phases were combined and filtered throughsodium sulfate. Solvent (about 600 mL) was removed by distillation atatmospheric pressure and isopropanol (250 mL) was added. Additionalsolvent (about 300 mL) was distilled at atmospheric pressure andisopropanol (250 mL) was added. Additional solvent (about 275 mL) wasdistilled at atmospheric pressure and isopropanol (250 mL) was added.The solution was cooled at about 25° C. with stirring and after about 12hours, the crystalline solid was filtered, washed with isopropanol anddried (116.5 g, 70%).

Synthesis of4-hydroxy-4-methyl-2-(4′-[2″-piperidino]-ethoxy)phenyl-3-(4′″-tetrahydropyranyloxy)phenyl-7-tetrahydropyranyloxy-chromane(10)

A solution of2-tetrahydropyranyloxy-4-hydroxy-2′-(4″-tetrahydropyranyloxyphenyl)acetophenone 4 (1 kg, 2.42 mole),4-[2-(1-piperidino)ethoxy]benzaldehyde 5 (594 g, 2.55 mole) (availablefrom Chemsyn Science Laboratories, Lenexa, Kans.) and piperidine (82.4g, 0.97 mole) (available from Aldrich Chemical Company Inc., Milwaukee,Wis.) in toluene (8 L) was refluxed under nitrogen with a Dean & Starkapparatus until one equivalent of water (44 mL) was collected. Toluene(6.5 L) was removed from the solution by distillation at atmosphericpressure. Dimethylformamide (6.5 L) and1,8-diazabicyclo[5,4,0]undec-7-ene (110.5 g, 0.726 mole) were added. Thesolution was agitated for about 8 hours at room temperature to isomerizethe chalcone 8 to chromanone 9 and then added to a mixture of water andice (8 L) and toluene (4 L). The phases were separated and the toluenelayer washed with water (5 L). The combined aqueous washes wereextracted with toluene (3×4 L). The combined toluene extracts werefinally washed with brine (3×4 L) concentrated at atmospheric pressureto 5.5 L and then cooled to −10° C. With continued external cooling andstirring under nitrogen, a 3M solution of methylmagnesium chloride inTHF (2.5 L, 7.5 mole) (available from Aldrich Chemical Company Inc.,Milwaukee, Wis.) was added, maintaining the temperature below 0° C.After all the Grignard reagent was added, the external cooling wasremoved and the mixture allowed warm to room temperature. The mixturewas stirred at this temperature for about 24 hours. The mixture wasagain cooled to about −20° C. and with continued external cooling andstirring, saturated ammonium chloride solution (200 mL) was addedslowly, maintaining the temperature below 20° C. The mixture was stirredfor 2 hours and then added the saturated ammonium chloride solution (2L) and toluene (4 L) and agitated for five minutes. The phases wereseparated and the aqueous layer extracted with toluene (2×4 L). Thecombined toluene extracts were washed with dilute hydrochloric aciduntil the solution became homogenous and then with brine (3×4 L). Thetoluene solution was finally concentrated at atmospheric pressure to 2L. This solution was used directly in the next step.

Synthesis of(2R,S)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-1-benzopyran(1S)-10-camphorsulphonic acid salt (±12)

To the toluene solution of4-hydroxy-4-methyl-2-(4′-[-2″-piperidino]-ethoxy)-phenyl-3-(4′″-tetrahydropyranyloxy)phenyl-7-tetrahydropyranyloxychromane (10) was added acetone (6 L), water (0.3 L) and(S)-10-camphorsulphonic acid (561 g, 2.42 mole) (available from AldrichChemical Company Inc. Milwaukee Wis.). The mixture was agitated undernitrogen for 48 hours after which time the solid(2R,S)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-1-benzopyran(1S)-10-camphor sulphonic acid salt (12) was filtered, washed withacetone and dried (883 g). This material was used in the next (HH) stepwithout further purification.

Synthesis of(2S)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-1-benzopyran(1S)-10-camphorsulphonic acid salt (13, (+)-EM-652(1S)-CSA salt)

A suspension of(2R,S)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-benzopyran(1S)-10-cannphorsulphonic acid salt ±12 (759 g) in 95% ethanol washeated with stirring to about 70° C. until the solid had dissolved. Thesolution was allowed to cool to room temperature with stirring thenseeded with a few crystals of(2S)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-[2′″-piperidino]ethoxy)phenyl)-2H-1-benzopyran(1S)-10-camphorsulphonic acid salt 13. The solution was stirred at roomtemperature for about three days in total. The crystals were filtered,washed with 95% ethanol and dried (291 g, 76%). The de of the productwas 94.2% and the purity 98.8%.

Synthesis of acolbifene((S)-(+)-7-hydroxy-3-(4′-hydroxyphenyl)-4-methyl-2-(4″-(2′″-piperidinoethoxy)phenyl)-2H-1-benzopyranhydrochloride, EM-01538, (EM-652.HCl))

A suspension of compound 13 (EM-652-(+)-CSA salt, 500 mg, 0.726 mmol) indimethylformamide (11 μL, 0.15 mmol) was treated with an 0.5 M aqueoussodium carbonate solution (7.0 mL, 3.6 mmol), and stirred for 15 min.The suspension was treated with ethyl acetate (7.0 mL) and stirredduring 4 h. The organic phase was then washed with an aqueous saturatedsodium carbonate solution (2×5 mL) and brine (1×5 mL) dried overmagnesium sulfate, and concentrated. A solution of the resulting pinkfoam (EM-652) in ethanol (2 mL) was treated with 2 N hydrochloric acid(400 μL, 0.80 mmol), stirred for 1 h, treated with distilled water (5mL), and stirred during 30 min. The resulting suspension was filtered,washed with distilled water (5 mL), dried in air and under high vacuum(65° C.) to give a creamy powder (276 mg, 77%): Fine off-white powder;Scanning calorimetry: Melting peak onset at 219° C., ΔH=83 J/g; [α]²⁴_(D)=154° in methanol 10 mg/mL; ¹H NMR (300 MHz, CD₃OD) δ(ppm) 1.6(broad, 2H, H-4′″), 1.85 (broad, 4H, H-3″″ and 5″″), 2.03 (s, 3H, CH₃),3.0 and 3.45 (broad, 4H, H-2″″ and 6″″), 3.47 (t, J=4.9 Hz, 2H, H-3′″),4.26 (t, J=4.9 Hz, 2H, H-2′″), 5.82 (s, 1H, H-2), 6.10 (d, J=2.3 Hz, 1H,H-8), 6.35 (dd, J=8.4, 2.43 Hz, 1H, H-6), 6.70 (d, J=8.6 Hz, 2H, H-3′,and H-5′), 6.83 (d, J=8.7 Hz, 2H, H-3″ and H-5″), 7.01 (d, J=8.5 Hz, 2H,H-2′ and H-6′), 7.12 (d, J=8.4 Hz, 1H, H-5), 7.24 (d, J=8.6 Hz, 2H, H-2″and H-6″); ¹³C RMN (CD₃OD, 75 MHz) δ ppm 14.84, 22.50, 23.99, 54.78,57.03, 62.97, 81.22, 104.38, 109.11, 115.35, 116.01, 118.68, 125.78,126.33, 130.26, 130.72, 131.29, 131.59, 134.26, 154.42, 157.56, 158.96,159.33. Elemental Composition: C, H, N, Cl: Theory; 70.51, 6.53, 2.84,7.18, %. Found: 70.31, 6.75, 2.65, 6.89%.

Example 2 Materials and Methods Animals

Female BALB/c mice (BALB/cAnNCrlBR) weighing 18-20 g were obtained fromCharles-River, Inc. (St-Constant, Quebec, Canada) and housed 5 per cagein a temperature (23±1° C.)- and light (12 h light/day, lights on at7:15)-controlled environment. The mice were fed rodent chow and tapwater ad libitum. The animals were ovariectomized (OVX) under Isofluraneanesthesia via bilateral flank incisions and randomly assigned to groupsof 10 animals. Ten mice were kept intact as controls.

Treatments

In the first experiment, the tested compounds (FIGS. 17 and 18), namelyEM-652.HCl (acolbifene), lasofoxifene (as free base; active and inactiveenantiomers) and raloxifene, were administered orally by gavage oncedaily at doses of 1, 3 or 10 μg/animal for 9 days, starting 2 days afterovariectomy. In the second experiment (Table 4), TSE 424 wasadministered orally by gavage once daily at doses of 1, 3, 10 or 30μg/animal for 9 days, starting 2 days after ovariectomy. In bothexperiments, to evaluate the antiestrogenic activity, treatment withestrone (E₁, 0.06 μg, s.c. injection, twice daily) was started 5 dayspost-ovariectomy and was administered for a 6 day-period. Compounds weredissolved in ethanol (4% final concentration) and administered in 0.4%methylcellulose. Mice in the intact and OVX control groups received thevehicle alone (4% ETOH-0.4% methylcellulose) during the 9-day period.The animals were killed by exsanguination at the abdominal aorta on the11^(th) morning following ovariectomy. The uteri and vagina were rapidlydissected, weighed, and kept in 10% buffered formalin for furtherhistologic examination.

Article 1: Results Experiment 1:

As illustrated in FIG. 17, EM-652.HCl (acolbifene) administered at thedaily oral doses of 1 μg, 3 μg, and 10 μg caused respective 24%, 48%,and 72% inhibitions of estrone-stimulated uterine weight (p<0.01 for alldoses versus control) while raloxifene administered at the same dosescaused respective 6% (NS), 14% (p<0.01) and 43% (p<0.01) inhibitions ofthis parameter. Lasofoxifene (as free base), on the other hand, had noinhibitory effect at the lowest dose used while it caused respective 25%(p<0.01) and 44% (p<0.01) inhibitions of estrone-stimulated uterineweight at the daily doses of 3 μg and 10 μg. The inactive enantiomer oflasofoxifene exerted no inhibitory effect on this parameter at any doseused.

When compounds were administered alone (in the absence of estrone) toovariectomized mice at the daily oral doses of 1 μg and 10 μg,EM-652.HCl had no significant stimulatory effect on uterine weight atboth doses used, while treatment with 10 μg of lasofoxifene andraloxifene caused respective 93% (p<0.01) and 85% (p<0.01) stimulationsof uterine weight (FIG. 18), thus indicating an estrogenic effect ofthese latter compounds on this parameter. Similarly, EM-652.HCl exertedno significant stimulatory effect on vaginal weight (FIG. 18) whileadministration of 10 μg of lasofoxifene and raloxifene caused respective73% (p<0.01) and 56% (p<0.01) stimulations of vaginal weight. On theother hand, the inactive enantiomer of lasofoxifene had no stimulatoryeffect on uterine and vaginal weight.

Experiment 2:

As shown in Table 4, TSE 424 administered at the daily oral doses of 1μg, 3 μg, 10 μg or 30 μg caused respective 12% (NS), 47%, 74%, and 94%inhibitions of estrone-stimulated uterine weight (p<0.01 for the threehighest doses versus E₁-control). When the compound was administeredalone (in the absence of estrone) to ovariectomized mice at the dailyoral doses of 3 μg and 30 μg, TSE 424 had no significant stimulatoryeffect on uterine weight at both doses used (Table 4).

TABLE 4 Effect on uterine weight of increasing concentrations of TSE 424administered orally for 9 days to ovariectomized mice simultaneouslytreated or not with estrone. **p < 0.01 versus E₁-treated control.UTERINE WEIGHT TREATMENT (mg) INTACT  54.6 ± 12.5** OVX  15.6 ± 1.3**OVX + E₁ 118.3 ± 6.0  OVX + E₁ + TSE 424 1 μg 105.5 ± 6.1  OVX + E₁ +TSE 424 3 μg  69.7 ± 4.4** OVX + E₁ + TSE 424 10 μg  42.1 ± 2.7** OVX +E₁ + TSE 424 30 μg  21.7 ± 1.7** OVX + TSE 424 3 μg 18.3 ± 1.2  OVX +TSE 424 30 μg 17.7 ± 1.6 

Example 3 Preventive Effects on Bone Loss, Serum Lipids and Total BodyFat Animals and Treatment

Ten to twelve week-old female Sprague-Dawley rats (Crl:CD(SD)Br)(Charles River Laboratory, St-Constant, Canada) weighing approximately220-270 g at start of treatment were used. The animals were acclimatizedto the environmental conditions (temperature: 22±3° C.; humidity:50±20%; 12-h light-12-h dark cycles, lights on at 07:15 h) for at least1 week before starting the experiments. The animals were housedindividually and were allowed free access to tap water and a pelletedcertified rodent feed (Lab Diet 5002, Ralston Purina, St-Louis, Mo.).Experiments were conducted in an animal facility approved by theCanadian Council on Animal Care (CCAC) and the Association forAssessment and Accreditation of Laboratory Animal Care (AAALAC) inaccordance with the CCAC Guide for Care and Use of Experimental Animals.

In a first experiment, one hundred fifty-four rats were randomlydistributed between 11 groups of 14 animals each as follows: 1) Intactcontrol; 2) OVX control; 3) OVX+E₂ (1 mg/kg); 4) OVX+EM-652.HCl (2.5mg/kg); 5) OVX+E₂+EM-652.HCl; 6) OVX+dehydroepiandrosterone (DHEA; 80mg/kg); 7) OVX+DHEA+EM-652.HCl; 8) OVX+DHEA+E₂; 9)OVX+DHEA+E₂+EM-652.HCl; 10) OVX+GW 5638; 11) OVX+E₂+GW 5638. On Day 1 ofthe study, the animals of the appropriate groups were bilaterallyovariectomized (OVX) under isoflurane anesthesia. The DHEA was appliedtopically on the dorsal skin as a solution in 50% ethanol-50% propyleneglycol while the other tested compounds were administered as suspensionin 0.4% methylcellulose by oral gavage. Treatments were initiated on Day2 of the study and were performed once daily during 3 months.

In the second experiment, one hundred thirty-two rats were randomlydistributed between 9 groups of 14 or 15 animals each as follows: 1)Intact control; 2) OVX control; 3) OVX+Premarin (0.25 mg/kg); 4)OVX+EM-652.HCl (2.5 mg/kg); 5) OVX+Premarin+EM-652.HCl; 6) OVX+TSE 424(2.5 mg/kg); 7) OVX+Premarin+TSE 424; 8) OVX+lasofoxifene (tartratesalt; racemate; 2.5 mg/kg); 9) OVX+Premarin+lasofoxifene. On Day 1 ofthe study, the animals of the appropriate groups were bilaterally OVXunder isoflurane anesthesia. Tested compounds were administered assuspension in 0.4% methylcellulose by oral gavage. Treatments wereinitiated on Day 2 of the study and were performed once daily during 26weeks. In both experiments, animals not receiving a test article weretreated with the appropriate vehicle alone during the same period.

Bone Mineral Density Measurements

After 3 months (experiment 1) or 26 weeks (experiment 2) of treatment,individual rats under Isoflurane anesthesia had their whole bodyskeleton and lumbar spine scanned using dual energy x-ray absorptiometry(DEXA; QDR 4500A, Hologic, Waltham, Mass.) and a Regional HighResolution Scan software. The bone mineral density (BMD) of the lumbarspine (vertebrae L2 to L4) and the total body composition (fatpercentage) were determined.

Serum Assays

After 3 months (experiment 1) or 26 weeks (experiment 2) of treatment,blood samples were collected at the jugular vein from overnight fastedanimals (under Isoflurane anesthesia). Samples were processed for serumpreparation and frozen at −80° C. until assay. Serum cholesterol levelsand alkaline phospatase activity (ALP) were determined using theBoehringer Mannheim Diagnostic Hitachi 911 Analyzer (Boehringer MannheimDiagnostic Laboratory Systems).

Statistical Analyses

Data are expressed as means±SEM. Statistical significance was determinedaccording to the multiple-range test of Duncan-Kramer (Kramer 1956).

Results

As shown in Table 5, 3 months after ovariectomy, BMD of the lumbar spinewas 10% lower in OVX control animals than in intact controls (p<0.01).At the doses used, the administration of estradiol and EM-652.HCl aloneprevented lumbar spine BMD loss by 98% (p<0.01) and 65% (p<0.05),respectively, while the combined treatment with E₂ and EM-652.HClprevented the OVX-induced decrease in lumbar spine BMD by 61% (p<0.05).On the other hand, while the administration of DHEA alone preventedlumbar spine BMD by 43% (p<0.05), the combined treatment withDHEA+E₂+EM-652.HCl prevented the OVX-induced decrease in lumbar spineBMD by 91% and led to BMD value not different from intact controls.

In Table 6, 26 weeks after ovariectomy, BMD of the lumbar spine was 18%lowered compared to intact controls (p<0.01). The administration ofPremarin, EM-652.HCl, TSE 424 and lasofoxifene alone prevented lumbarspine BMD by 54%, 62%, 49% and 61%, respectively (all p<0.01 versus OVXcontrols). The addition of Premarin to EM-652.HCl, TSE 424 orlasofoxifene led to lumbar spine BMD values not significantly differentfrom those obtained with the administration of each SERM alone (Table6). Similarly, the addition of DHEA to E₂ or to EM-652.HCl completelyprevented the OVX-induced decrease in lumbar spine BMD (Table 5). Thepositive effect of DHEA on BMD is also supported by its effect on serumalkaline phosphatase activity (ALP), a marker of bone formation andturnover. ALP activity was increased from 73±6 IU/L in OVX controlanimals to 224±18 IU/L, 290±27 IU/L, 123±8 IU/L and 261±20 IU/L (allp<0.01) in DHEA-, DHEA+EM-652.HCl-, DHEA+E₂- andDHEA+E₂+EM-652.HCl-treated animals, respectively, thus suggesting astimulatory effect of DHEA on bone formation (Table 7).

In addition to the preventive effects on bone loss, the administrationof EM-652.HCl, TSE 424, lasofoxifene, GW 5638, DHEA and E₂ exerts somebeneficial effects on total body fat percentage and serum lipids. Afterthree months of ovariectomy, total body fat was increase by 22% (p<0.05;Table 7). The administration of EM-652.HCl completely prevented theOVX-induced fat percentage increase while the addition of DHEA and/or E₂to the SERM led to fat percentage values below those observed in intactcontrol animals. After 26 weeks of ovariectomy, the 40% fat increaseinduced by estrogen deficiency was reversed by 74%, 78%, 75% and 114%following the administration of Premarin, EM-652.HCl, TSE 424 orlasofoxifene, respectively, while the addition of Premarin to each SERMcompletely prevented the OVX-induced fat percentage increase (Table 8).

As shown in Table 7, three months after ovariectomy, a 22% increase inserum cholesterol levels was observed in OVX control rats compared tointact controls (p<0.01). In fact, serum cholesterol was increased from2.01±0.11 mmol/L in intact animals to 2.46 V 0.08 mmol/L in OVXcontrols. The administration of E₂ or DHEA alone decrease serumcholesterol levels to 1.37±0.18 mmol/L and 1.59±0.10 mmol/L,respectively, while the administration of EM-652.HCl alone or incombination with E₂ and/or DHEA led to cholesterol levels significantlylower (between 0.65 to 0.96 mmol/L) than those found in intact animals(2.01±0.11 mmol/L). Similarly, the administration of GW 5638, TSE 424and lasofoxifene alone or in combination with E₂ or Premarin completelyprevented the OVX-induced increase on serum cholesterol levels and ledto values lower than those found in intact animals (Tables 7 and 8).

TABLE 5 EFFECT ON PREVENTION OF BONE LOSS FOLLOWING 3 MONTH-TREATMENTWITH ESTRADIOL, EM-652.HCl, GW 5638 OR DHEA, ADMINISTERED ALONE OR INCOMBINATION, TO OVARIECTOMIZED FEMALE RATS ARTICLE III. LUMBAR SPINEPrevention ARTICLE II. BMD of Bone ARTICLE IV. TREATMENT (g/cm²) Loss(%) 1) Intact   0.2461 ± 0.0049** 100 OVX 0.2214 ± 0.0044 — OVX + E₂  0.2457 ± 0.0049** 98 OVX + EM-652.HCl  0.2374 ± 0.0027* 65 OVX +EM-652.HCl + E₂  0.2364 ± 0.0037* 61 Section 1.02 OVX + DHEA 0.2321 ±0.0034 43 Section 1.03 OVX + DHEA +  0.2458 ± 0.0037** 99 EM 652.HClSection 1.04 OVX + DHEA + E₂  0.2496 ± 0.0029** 114 Section 1.05 OVX +DHEA + E₂ +  0.2439 ± 0.0043** 91 EM-652.HCl Section 1.06 OVX + GW 56380.2299 ± 0.0060 34 Section 1.07 OVX + GW 5638 + E₂ 0.2344 ± 0.0054 53 *,p < 0.05; **, p < 0.01, experimental versus OVX control rats.

TABLE 6 EFFECT ON PREVENTION OF BONE LOSS FOLLOWING 26 WEEK- TREATMENTWITH PREMARIN, EM-652.HCl, TSE 424 OR LASOFOXIFENE, ADMINISTERED ALONEOR IN COMBINATION WITH PREMARIN, TO OVARIECTOMIZED FEMALE RATS ARTICLEVI. LUMBAR SPINE Prevention of ARTICLE V. BMD Bone Loss ARTICLE VII.TREATMENT (g/cm2) (%) 1) Intact  0.2482 ± 0.0067** 100 OVX 0.2035 ±0.0035 — OVX + Premarin  0.2277 ± 0.0028** 54 OVX + EM-652.HCl  0.2311 ±0.0040** 62 OVX + Premarin + EM-652.HCl  0.2319 ± 0.0057** 64 Section1.08 OVX + TSE 424  0.2252 ± 0.0058** 49 Section 1.09 OVX + Premarin +TSE 424  0.2223 ± 0.0046** 42 Section 1.10 OVX + lasofoxifene  0.2307 ±0.0040** 61 Section 1.11 OVX + Premarin +  0.2357 ± 0.0035** 72lasofoxifene **, p < 0.01, experimental versus OVX control rats.

TABLE 7 EFFECT ON TOTAL BODY FAT PERCENTAGE, SERUM CHOLESTEROL LEVELSAND ALKALINE PHOSPHATASE ACTIVITY FOLLOWING 3 MONTH-TREATMENT WITHESTRADIOL, EM-652.HCl, GW 5638 OR DHEA, ADMINISTERED ALONE OR INCOMBINATION, TO OVARIECTOMIZED FEMALE RATS ARTICLE ARTICLE X ARTICLEARTICLE VIII IX TOTAL CHOLES- XI ARTICLE XII FAT TEROL ALP TREATMENT (%)(mmol/L) (IU/L) 1) Intact 24.0 ± 1.5*  2.01 ± 0.11**  39 ± 2** OVX 29.2± 1.5  2.46 ± 0.08 73 ± 6  OVX + E₂  19.5 ± 2.5**  1.37 ± 0.18** 59 ± 4 OVX + EM-652.HCl  23.2 ± 1.4**  0.87 ± 0.04** 91 ± 6* OVX + EM-652.HCl +E₂  20.4 ± 1.4**  0.96 ± 0.07** 92 ± 5* Section 1.12 OVX + DHEA  17.3 ±1.5**  1.59 ± 0.10**  224 ± 18** Section 1.13 OVX +  18.0 ± 1.1**  0.65± 0.06**  290 ± 27** DHEA + EM-652.HCl Section 1.14 OVX +  15.8 ± 1.3** 1.08 ± 0.08** 123 ± 8** DHEA + E₂ Section 1.15 OVX +  19.2 ± 1.6** 0.71 ± 0.08**  261 ± 20** DHEA + E₂ + EM-652.HCl Section 1.16 OVX + GW5638  21.9 ± 1.4**  1.14 ± 0.08** 72 ± 6  Section 1.17 OVX +  23.2 ±1.2**  0.91 ± 0.07** 80 ± 6  GW 5638 + E₂ *, p < 0.05; **, p < 0.01,experimental versus OVX control rats.

TABLE 8 EFFECT ON TOTAL BODY FAT PERCENTAGE, SERUM CHOLESTEROL LEVELSAND ALKALINE PHOSPHATASE ACTIVITY FOLLOWING 26 WEEK-TREATMENT WITHPREMARIN, EM-652.HCl, TSE 424 OR LASOFOXIFENE, ADMINISTERED ALONE OR INCOMBINATION WITH PREMARIN, TO OVARIECTOMIZED FEMALE RATS ARTICLE ARTICLEXV ARTICLE ARTICLE XIII XIV CHOLES- XVI ARTICLE XVII TOTAL FAT TEROL ALPTREATMENT (%) (mmol/L) (IU) 1) Intact  25.5 ± 1.8**  2.11 ± 0.11** 33 ±2* OVX 35.7 ± 1.6  2.51 ± 0.09 60 ± 6  OVX + Premarin  28.2 ± 1.8** 1.22 ± 0.07** 49 ± 3  OVX + EM-652.HCl  27.7 ± 1.4**  0.98 ± 0.06** 78± 4  OVX + EM-652.HCl +  25.7 ± 2.2**  1.10 ± 0.07** 81 ± 6  PremarinSection 1.18 OVX +  28.0 ± 1.8**  1.15 ± 0.05** 85 ± 6  TSE 424 Section1.19 OVX +  25.7 ± 1.7**  1.26 ± 0.14**  98 ± 22** TSE 424 + PremarinSection 1.20 OVX +  24.1 ± 1.3**  0.60 ± 0.02** 116 ± 9** lasofoxifeneSection 1.21 OVX +  23.8 ± 1.9**  0.81 ± 0.12** 107 ± 6** lasofoxifene +Premarin *, p < 0.05; **, p < 0.01, experimental versus OVX controlrats.

Example 4 Preventive Effects on Bone Loss Following Treatment with theSERMs Em-652.HCl (Acolbifene), TSE-424 and ERA-923, Administered Aloneand in Combination with DHEA to Ovariectomized Female Rats Animals andTreatment

Ten to twelve week-old female Sprague-Dawley rats (Crl:CD(SD)Br)(Charles River Laboratory, St-Constant, Canada) weighing approximately220-270 g at start of treatment were used. The animals were acclimatizedto the environmental conditions (temperature: 22±3° C.; humidity:50±20%; 12-h light-12-h dark cycles, lights on at 07:15 h) for at least1 week before starting the experiments. The animals were housedindividually and were allowed free access to tap water and a pelletedcertified rodent feed (Lab Diet 5002, Ralston Purina, St-Louis, Mo.).Experiments were conducted in an animal facility approved by theCanadian Council on Animal Care (CCAC) and the Association forAssessment and Accreditation of Laboratory Animal Care (AAALAC) inaccordance with the CCAC Guide for Care and Use of Experimental Animals.

One hundred twenty-six rats were randomly distributed between 9 groupsof 14 animals each as follows: 1) Intact control; 2) OVX control; 3)OVX+EM-652.HCl (2.5 mg/kg); 4) OVX+TSE-424 (EM-4803, 2.5 mg/kg); 5)OVX+ERA-923 (EM-3527, 2.5 mg/kg); 6) OVX+dehydroepiandrosterone (DHEA;80 mg/kg); 7) OVX+DHEA+EM-652.HCl; 8) OVX+DHEA+TSE-424; 9)OVX+DHEA+ERA-923. On Day 1 of the study, the animals of the appropriategroups were bilaterally ovariectomized (OVX) under isofluraneanesthesia. The DHEA was applied topically on the dorsal skin as asolution in 50% ethanol-50% propylene glycol while the tested SERMs wereadministered as suspension in 0.4% methylcellulose by oral gavage.Treatments were initiated on Day 2 of the study and were performed oncedaily during 5 weeks.

Bone Mineral Density Measurements

After 5 weeks of treatment, individual rats under Isoflurane anesthesiahad their lumbar spine, femur and tibia scanned using dual energy x-rayabsorptiometry (DEXA; QDR 4500A, Hologic, Waltham, Mass.) and a RegionalHigh Resolution Scan software. The bone mineral density (BMD) of thelumbar spine (vertebrae L2 to L4), distal femoral metaphysis (DFM) andproximal tibial metaphysis (PTM) were determined.

Statistical Analyses

Data are expressed as means±SEM. Statistical significance was determinedaccording to the multiple-range test of Duncan-Kramer {Kramer, 1956#37421.

Results

As shown in Table 9, after 5 weeks of ovariectomy, BMD of the lumbarspine was 9% lower in Ovx control animals than in intact controls. Atthe dose used the administration of the SERMs: EM-652.HCl, TSE-424 orERA-923 alone prevented lumbar spine BMD loss by 86%, 53% and 78%,respectively. On the other hand, the administration of DHEA aloneprevented lumbar spine BMD loss by 44%, while the combined treatmentwith DHEA+EM-652.HCl, DHEA+TSE-424 or DHEA+ERA-923 prevented theOVX-induced decrease in lumbar spine BMD by 94%, 105% and 105%,respectively.

Bone mineral density of the distal femoral metaphysis (DFM) wasdecreased by 10% after 5 weeks of ovariectomy (Table 9). Theadministration of the SERMs: EM-652.HCl, TSE-424 or ERA-923 aloneprevented DFM BMD loss by 95%, 70% and 83%, respectively. On the otherhand, the administration of DHEA alone prevented DFM BMD loss by 71%,while the combined treatment with DHEA+EM-652.HCl, DHEA+TSE-424 orDHEA+ERA-923 completely prevented the OVX-induced decrease in DFM BMDand led to DFM BMD values higher than those observed in intact controlanimals. Similar results were obtained on proximal tibial metaphysis BMD(Table 9).

TABLE 9 EFFECT ON PREVENTION OF BONE LOSS FOLLOWING 5 WEEK-TREATMENTWITH THE SERMs EM-652.HCI, TSE-424 AND ERA-923, ADMINISTERED ALONE OR INCOMBINATION WITH DHEA, TO OVARIECTOMIZED FEMALE RATS DISTAL FEMORALLUMBAR SPINE METAPHYSIS (DFM) PROXIMAL TIBIAL METAPHYSIS (L2-L4)Prevention (PFM) BMD Prevention of BMD of Bone BMD Prevention of BoneTREATMENT (g/cm²) Bone Loss (%) (g/cm²) Loss (%) (g/cm²) Loss (%) Intact0.2261 ± 0.0046 100  0.3024 ± 0.0040 100  0.2828 ± 0.0032 100  Ovx0.2051 ± 0.0037 — 0.2709 ± 0.0036 — 0.2560 ± 0.0028 — Ovx + EM-652.HCI0.2232 ± 0.0031 86 0.3008 ± 0.0055 95 0.2806 ± 0.0035 92 Ovx + TSE-4240.2162 ± 0.0035 53 0.2929 ± 0.0042 70 0.2750 ± 0.0039 71 Ovx + ERA-9230.2214 ± 0.0029 78 0.2969 ± 0.0029 83 0.2805 ± 0.0034 91 Ovx + DHEA0.2144 ± 0.0028 44 0.2934 ± 0.0046 71 0.2672 ± 0.0041 42 Ovx + DHEA +EM- 0.2249 ± 0.0023 94 0.3122 ± 0.0045 131  0.2867 ± 0.0047 115  652.HCIOvx + DHEA + TSE-424 0.2271 ± 0.0030 105  0.3099 ± 0.0040 124  0.2833 ±0.0034 102  Ovx + DHEA + ERA-923 0.2271 ± 0.0030 105  0.3072 ± 0.0053115  0.2817 ± 0.0034 96

Example 5 Effect of Compounds of the Invention on Alkaline PhosphataseActivity in Human Endometrial Adenocarcinoma Ishikawa Cells MaterialsMaintenance of Stock Cell Cultures

The human Ishikawa cell line derived from a well differentiatedendometrial adenocarcinoma was kindly provided by Dr. Erlio Gurpide, TheMount Sinai Medical Center, New York, N.Y. The Ishikawa cells wereroutinely maintained in Eagle's Minimum Essential Medium (MEM)containing 5% (vol/vol) FBS (Fetal Bovine Serum) and supplemented with100 U/mL penicillin, 100 μg/mL streptomycin, 0.1 mM non-essential aminoacids solution. Cells were plated in Falcon T75 flasks at a density of1.5×10⁶ cells at 37° C.

Cell Culture Experiments

Twenty four hours before the start of the experiment, the medium of nearconfluent Ishikawa cells was replaced by fresh estrogen-free basalmedium (EFBM) consisting of a 1:1 (v:v) mixture of phenol red-free Ham'sF-12 and Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 100U/mL penicillin, 100 μg/mL streptomycin, 2 mM glutamine, and 5% FBStreated twice with dextran-coated charcoal to remove endogenoussteroids. Cells were then harvested by 0.1% pancreatin (Sigma) and 0.25mM HEPES, resuspended in EFBM and plated in Falcon 96, wellflat-bottomed microtiter plates at a density of 2.2×10⁴ cells/well in avolume of 100 μl and allowed to adhere to the surface of the plates for24 h. Thereafter, medium was replaced with fresh EFBM containing theindicated concentrations of compounds in a final volume of 200 μl. Cellswere incubated for five days, with a medium change after 48 h.

Alkaline Phosphatase Assay

At the end of the incubation period, microtiter plates were inverted andgrowth medium was decanted. The plates were rinsed with 200 μl by wellof PBS (0.15 M NaCl, 10 mM sodium phosphate, pH 7.4). PBS was thenremoved from the plates while carefully leaving some residual PBS, andthe wash procedure was repeated once. The buffered saline was thendecanted, and the inverted plates were blotted gently on a paper towel.Following replacement of the covers, the plates were placed at −80° C.for 15 min followed by thawing at room temperature for 10 min. Theplates were then placed on ice, and 50 μl of an ice-cold solutioncontaining 5 mM p-nitrophenyl phosphate, 0.24 mM MgCl₂, and 1 Mdiethanolamine (pH 9.8) were added. Plates were then warmed to roomtemperature, and the yellow color from the production of p-nitrophenylwas allowed to develop (8 min). Plates were monitored at 405 nm in anenzyme-linked immunosorbent assay plate reader (BIO-RAD, model 2550 EIAReader).

Calculations

Dose-response curves as well as IC₅₀ values were calculated using aweighted iterative nonlinear squares regression.

TABLE 10 Maximal Inhibition of Maximal stimulation 1 nM E₂- inhibitionof induced of 1 nM E₂- alkaline stimulation induced phosphatase ofalkaline stimulation % of 1 nM E₂ phosphatase of alkaline stimulation *IC₅₀ (nM) phosphatase CODE (nb of (nb of (nb of NAME NAME STRUCTUREexperiments) experiments) experiments) EM-652.HCl (acolbifene)EM-652.HCl; (EM-1538)

1.88 ± 0.26 (22)  1.52 ± 0.22 (18)  98.97 ± 0.174 (18)  OH- toremifeneEM-880

29.6 ± 2.1  (6) 72.1 ± 7.6  (3) 75.73 ± 3.52  (3) GW-5638 EM-1796

7.75 ± 5.5  (2) No inhibition raloxifene LY 156758 EM-1105

12.8 ± 1.7  (8) 3.39 ± 0.9  (6) 94.31 ± 1.74  (5) LY 353381 EM-1665

15.5 ± 0.25 (5) 1.87 ± 0.07 (2) 90.25 ± 0.127 (2) lasofoxifene (freebase) EM-3114

17.9 (1) 4.24 (1)  85.14 (1) ERA-923 EM-3527

 0.6 (1) 5.84 (1) 100.16 (1) * % of 1 nM E₂ stimulation = OD 405 nmcompound-OD 405 nm basal/OD 405 nm 1 nM E₂-OD 405 nm basal Please seealso Labrie et al. 1999.

Example 6 Effect of EM-652.HCl (Acolbifene), TSE 424, and Lasofoxifeneon the Proliferation of Human Breast Cancer MCF-7 Cells Methods:Maintenance of Stock Cell Cultures

MCF-7 human breast cancer cells were obtained from the American TypeCulture Collection # HTB 22 at passage 147 and routinely grown in phenolred-free Dulbecco's Modified Eagle's-Ham's F12 medium, the supplementsmentioned above and 5% FBS. The MCF-7 human breast adenocarcinoma cellline was derived from the pleural effusion of a Caucasian 69-year-oldfemale patient. MCF-7 cells were used between passages 148 and 165 andsubcultured weekly.

Cell Proliferation Studies

Cells in their late logarithmic growth phase were harvested with 0.1%pancreatin (Sigma) and resuspended in the appropriate medium containing50 ng bovine insulin/mL and 5% (v/v) FBS treated twice withdextran-coated charcoal to remove endogenous steroids. Cells were platedin 24-well Falcon plastic culture plates (2 Cm²/well) at the indicateddensity and allowed to adhere to the surface of the plates for 72 h.Thereafter, medium was replaced with fresh medium containing theindicated concentrations of compounds diluted from 1000× stock solutionsin 99% redistilled ethanol in the presence or absence of E₂. Controlcells received only the ethanolic vehicle (0.1% EtOH,v/v). Cells wereincubated for the specified time intervals with medium changes at 2- or3-day intervals. Cell number was determined by measurement of DNAcontent.

Calculations and Statistical Analysis

Dose-response curves as well IC₅₀ values were calculated using aweighted iterative nonlinear least-squares regression. All results areexpressed as means±SEM.

TABLE 11 Experiment 1 Maximal stimulation of DNA Inhibition of 1nM E₂ bytested stimulation of DNA compounds by tested % of 1nM E₂ compounds NAMECODE NAME stimulation * IC₅₀ (nM) EM-652.HCl EM-652.HCl; N.S. 0.796EM-1538 TSE 424 EM-4803 N.S. 3.68 Experiment 2 Stimulation of Inhibitionof 1nM E₂ DNA by tested stimulation of DNA compounds by tested % of 1nME₂ compounds NAME CODE NAME stimulation * IC₅₀ (nM) EM-652.HClEM-652.HCl; N.S. 0.205 EM-1538 lasofoxifene EM-3114 N.S. 0.379 (freebase)

Example 7 Comparison of the Effects of EM-652.HCl (Acolbifene),Tamoxifen, Toremifene, Droloxifene, Idoxifene, GW-5638, and Raloxifeneon the Growth of Human RZ-75-1 Breast Tumors in Nude Mice

The objective of this example was to compare the agonistic andantagonistic effects of EM-652.HCl and six other oral antiestrogens(SERMs) on the growth of the well-characterized estrogen-sensitiveZR-75-1 breast cancer xenografts in ovariectomized nude mice.

Materials and Methods Human ZR-75-1 Breast Cancer Cells

ZR-75-1 human breast cancer cells were obtained from the American TypeCulture Collection (Rockville, Md.) and cultured in phenol red-freeRPMI-1640 medium. The cells were supplemented with 2 mM L-glutamine, 1mM sodium pyruvate, 100 IU penicillin/mL, 100 μg streptomycin/mL, and10% (v/v) fetal bovine serum and incubated under an humidifiedatmosphere of 95% air/5% CO₂ at 37° C. Cells were passaged weekly andharvested at 85-90% confluence using 0.083% pancreatin/0.3 mM EDTA.

Animals and Tumor Inoculation

Homozygous female nu/nu Br athymic mice (28- to 42-day old) wereobtained from Charles River, Inc. (Saint-Constant, Québec, Canada). Themice (5 per cage) were housed in vinyl cages equipped with air filterlids, which were kept in laminar airflow hoods and maintained underpathogen-limiting conditions. The photoperiod was 12 hours of light and12 hours of darkness (lights on at 07:15). Cages, bedding and food(Agway Pro-Lab R-M-H Diet #4018) were autoclaved before use. Water wasautoclaved and provided ad libitum. Bilateral ovariectomy was performedunder isoflurane-induced anesthesia. At the time of ovariectomy, animplant of estradiol (E₂) was inserted subcutaneously to stimulateinitial tumor growth. E₂ implants were prepared in 1 cm-long Silastictubing (inside diameter: 0.062 inch; outside diameter: 0.095 inch)containing 0.5 cm of a 1:10 (w/w) mixture of estradiol and cholesterol.One week after ovariectomy, 2×10 6 ZR-75-1 (passage 93) cells wereinoculated subcutaneously in 0.1 mL of RPMI-1640 medium+30% Matrigel onboth flanks of each ovariectomized (OVX) mouse through a 2.5-cm-long22-gauge needle. After four weeks, the E₂ implants were replaced in allanimals by estrone-containing implants of the same size (E₁:chol, 1:25,w:w). Randomization and treatments were started one week later.

Treatments

One day prior to initiation of treatments, 255 mice bearing ZR-75-1tumors of an average area of 24.4±0.4 mm² (range 5.7 to 50.7 mm²) wererandomly assigned to 17 groups (with respect to tumor size), eachcontaining 15 mice (total of 29 or 30 tumors). The 17 groups includedtwo control groups (OVX and OVX+Estrone), seven groups supplemented withan estrone implant and treated with an antiestrogen and eight othergroups that received an antiestrogen alone. The estrone implants werethen removed from the animals in the ovariectomized control group (OVX)and in groups that were to receive the antiestrogen alone.Estrone-containing implants in the nine other groups were changedthereafter every 6 weeks. EM-652.HCl, raloxifene, droloxifene, idoxifeneand GW 5638 were synthesized in the medicinal chemistry division of theOncology and Molecular Endocrinology Research Center. tamoxifen waspurchased from Plantex (Netanya, Israel) while toremifene citrate waspurchased from Orion (Espoo, Finland). Under estrone stimulation, theantiestrogens were given at the daily oral dose of 50 μg (2 mg/kg, onaverage) suspended in 0.2 mL of 0.4% (w/v) methylcellulose. In theabsence of estrone stimulation, animals were treated with 200 μg (8mg/kg on average) of each antiestrogen once daily by the oral route.Animals in both control groups received 0.2 mL of the vehicle alone. Theantiestrogen suspensions at the appropriate concentration were preparedeach month, stored at 4° C. and used under constant agitation. Powderstock were hermetically stored at 4° C. (idoxifene, raloxifene,toremifene, GW 5638, droloxifene) or at room temperature (tamoxifen,EM-652.HCl).

Tumor Measurements and Necropsy

Two perpendicular diameters were recorded and tumor area (mm²) wascalculated using the formula: L/2×W/2×π. The area measured on the firstday of treatment was taken as 100%.

After 161 days of treatment, the remaining animals were anesthetizedwith isoflurane and killed by exsanguination. To further characterizethe effect of the estrogen and antiestrogens, estrogen-responsivetissues, such as the uterus and vagina, were immediately removed, freedfrom connective and adipose tissue and weighed. The uteri were preparedto evaluate endometrial thickness by image analysis performed with ImagePro-Plus (Media Cybernetics, Md., USA). In brief, uteri were fixed in10% formalin and embedded in parafin. Hematoxylin- and eosin-stainedsections of mice uteri were analyzed. Four images per uterus (2 peruterine horn) were analyzed. Mean epithelial cell height was measured inall animals of each group.

Response Criteria

Tumor response was assessed at the end of the study or at death of eachanimal, if it occurred during the course of the experiment. In thiscase, only data of mice that survived for at least half of the study (84days) were used in the tumor response analysis. In brief, completeregression identifies those tumors that were undetectable at the end ofthe experiment; partial regression corresponds to the tumors thatregressed ≧50% of their original size; stable response refers to tumorsthat regressed <50% or progressed ≦50%; and progression refers to tumorsthat progressed ≧0.50% compared with their original size.

Statistical Analyses

The change in total tumors surface areas between Day 1 and Day 161 wereanalyzed according to an ANOVA for repeated measurements. The modelincluded the treatment, time, and time-treatment interaction effectsplus the term to account for the strata at randomization. Thesignificance of the different treatments effects at 161 days was thustested by the time-treatment interaction. Analysis of the residualsindicated that the measurements on the original scale were not fittedfor analysis by an ANOVA nor any of the transformations that were tried.The ranks were therefore selected for the analyses. The effect of thetreatments on the epithelial thickness was assessed by a one-way ANOVAincluding also the strata at randomization. A posteriori pairwisecomparisons were performed using least square means statistics. Theovervall type 1 error rate (a) was controlled at 5% to declaresignificance of the differences. All calculations were performed usingProc MIXED on the SAS Software (SAS Institute, Carry, N.C.).

Results Antagonistic Effects on ZR-75-1 Tumor Growth

Estrone alone (OVX+E₁) caused a 707% increase in ZR-75-1 tumor sizeduring the 23 week-treatment period (FIG. 19). Administration of thepure antiestrogen EM-652.HCl (acolbifene) at the daily oral dose of 50μg to estrone-stimulated mice completely prevented tumor growth. Infact, not only tumor growth was prevented but after 23 weeks oftreatment, tumor size was 26% lower than the initial value at start oftreatment (p<0.04). This value obtained after treatment with EM-652.HClwas not statistically different from that observed after ovariectomyalone (OVX) where tumor size decreased by 61% below initial tumor size.At the same dose (50 μg) and treatment period, the six otherantiestrogens did not decrease initial average tumor size. Tumors inthese groups were all significantly higher than the OVX control groupand to the EM-652.HCl-treated group (p<0.01). In fact, compared topretreatment values, 23 weeks of treatment with droloxifene, toremifene,GW 5638, raloxifene, tamoxifen and idoxifene led to average tumor sizes478%, 230%, 227%, 191%, 87% and 86% above pretreatment values,respectively (FIG. 19).

Agonistic Effects on ZR-75-1 Tumor Growth

After 161 days of treatment with a daily dose of 200 μg of tamoxifen, inthe absence of estrone supplementation, the average tumor size increasedto 196% over baseline (p<0.01 vs OVX) (FIG. 20). On the other hand, theaverage tumor size of mice treated with idoxifene increased (125%)(p<0.01) while tumor size in mice treated with toremifene increased by86% (p<0.01) (FIG. 20). The addition of 200 μg of EM-652.HCl to 200 μgof tamoxifen completely inhibited the proliferation observed withtamoxifen alone (FIG. 15). On the other hand, treatment with EM-652.HCl(p=0.44), raloxifene (p=0.11), droloxifene (p=0.36) or GW 5638 (p=0.17)alone did not significantly change ZR-75-1 tumor size compared to theOVX control group, at the end of the experiment (FIG. 20).

Effects of Antiestrogens on Thickness of Uterine Epithelial Cells

The height of the endometrial epithelial cells was measured as the mostdirect parameter of agonistic and antagonistic effect of each compoundin the endometrium.

Effect of Daily 50 μg of Antiestrogen in the Presence of EstroneStimulation on Thickness of Uterine Epithelial Cells

At the daily oral dose of 50 μg, EM-652.HCl (acolbifene) inhibited thestimulatory effect of estrone on epithelial height by 70%. The efficacyof the six other antiestrogens tested were significantly lower (p<0.01).In fact, droloxifene, GW 5638, raloxifene, tamoxifen, toremifene andidoxifene inhibited estrone stimulation by 17%, 24%, 26%, 32%, 41% and50%, respectively (Table 12).

Effect of Daily 200 μg of Antiestrogen in Absence of Estrone Stimulationon Thickness of Uterine Epithelial Cells

In the absence of estrone stimulation, EM-652.HCl and droloxifene werethe only compounds tested that did not significantly increase the heightof epithelial cells (114% and 101% of the OVX control group value,respectively). Tamoxifen (155%), toremifene (135%) and idoxifene (176%)exerted a significant stimulation of uterine epithelial height (p<0.01vs OVX control group). Raloxifene (122%) and GW 5638 (121%) also exerteda statistically significant stimulation of uterine epithelial height(p<0.05 vs OVX control group (Table 12). The agonistic and antagonisticeffects of each antiestrogen measured on uterine and vaginal weight werein accordance with the pattern observed on uterine epithelium thickness(Data not shown).

TABLE 12 ENDOMETRIAL EPITHELIUM THICKNESS GROUP n (μm) ± SEM OVX CONTROL14 18.31 ± 0.04 OVX + E₁ + CONTROL 8 40.58^(b,d) ± 0.63   OVX + E₁ +EM-652•HCl 14 25.06^(b) ± 0.07  OVX + E₁ + TAMOXIFEN 10 33.44^(b,d) ±0.04   OVX + E₁ + TOREMIFENE 13 31.47^(b,d) ± 0.04   OVX + E₁ +RALOXIFENE 12 34.72^(b,d) ± 0.06   OVX + E₁ + DROLOXIFENE 12 36.71^(b,d)± 0.12   OVX + E₁ + IDOXIFENE 12 29.35^(b,d) ± 0.05   OVX + E₁ + GW 563812 35.30^(b,d) ± 0.07   OVX + EM-652•HCl 12 20.79 ± 0.10 OVX + TAMOXIFEN11 28.47^(b,d) ± 0.05   OVX + EM-652•HCl + 13 27.95^(b,d) ± 0.06  TAMOXIFEN OVX + TOREMIFENE 13 24.75^(b,c) ± 0.04   OVX + RALOXIFENE 1222.33^(a) ± 0.05  OVX + DROLOXIFENE 13 18.50 ± 0.07 OVX + IDOXIFENE 1132.14^(b,d) ± 0.05   OVX + GW 5638 13 22.22^(a) ± 0.05 ^(a,b)Experimental versus OVX control mice: ^(a)P < 0.05; ^(b)P < 0.01.^(c,d)Experimental versus EM-652•HCl treated-mice: ^(c)P < 0.05; ^(d)P <0.01.

Example 8 Radioactivity in the Brain of Female Rats Following a SingleOral Dose of ¹⁴C-EM-800 (20 Mg/Kg)

Example 8 shows the radioactivity in brain of rats following single oraldose of ¹⁴C-EM-800 (20 mg/kg), a SERM of the present invention. Forcomparison purposes, values for the blood, plasma, liver and uterus fromeach of these animals were included (Table 14). Tissue Distribution andExcretion of Radioactivity Following a Single Oral Dose of ¹⁴C-EM-800(20 ring/2 mL/kg) to Male and Female Long-Evans Rats. These numbersindicate that the amount of total drug-derived radioactivity in thebrain of female Long-Evans rats was very low (ng equiv/g tissue) and wasnot detected after 12 hr post dose. At 2 hours, radioactivity in thebrain was 412 lower than in liver, 21 times lower than in the uterus,8.4 times lower that in the blood and 13 times lower than in plasma.Since an unknown proportion of total brain radioactivity is due tocontamination by blood radioactivity, the values shown in Table 13 forbrain radioactivity are an overestimate of the level of ¹⁴C(EM-800)—related radioactivity in the brain tissue itself. Such datasuggest that the level of the antiestrogen in the brain tissue is toolow, to counteract the effect of exogenous estrogen. It is important tonote that some of the radioactivity detected in the brain tissue may bedue to residual blood in the tissue (Table 14). Additionally, theradiochemical purity of the ¹⁴C-EM-800 used for this study was minimally96.25%.

TABLE 13 Mean Concentration of Drug-Derived Radioactivity (ng EM-800equiv/g tissue) in Selected Tissues of Female Long-Evans Rats Followinga Single Oral Dose of ¹⁴C-EM-800 (20 mg/kg)^(a) Brain Blood Plasma TimeMean^(b) Mean^(b) Mean^(b) (hr) (% CV) (% CV) (% CV) 2 17.6 (29) 148.7(22) 224.6 (20) 4 17.1 (29) 66.9 (45) 103.2 (39) 6 15.6 (8) 48.3 (29)74.1 (31) 8 16.8 (31) 41.1 (12) 64.1 (14) 12 10.0^(c) (87) 28.7 (54)40.7 (55) 24 0 (NC) 4.7^(d) (173) 10.1 (86) 36 0 (NC) 0 (NC) 0 (NC) 48 0(NC) 0 (NC) 0 (NC) 72 0 (NC) 0 (NC) 0 (NC) 96 0 (NC) 0 (NC) 0 (NC) 168 0(NC) 0 (NC) 0 (NC) ^(a)Values from report tables for LREM 1129 (EM-800:Tissue Distribution and Excretion of Radioactivity Following a SingleOral Dose of ¹⁴C-EM-800 (20 mg/2 mL/kg) to Male and Female Long-EvansRats). ^(b)Limit of quantification (LOQ) of 1.2 ng EM-800 equivalent.^(c)One sample below the LOQ; 0 used in calculation of mean. ^(d)Twosamples below the LOQ; 0 used in calculation of mean. % CV: Coefficientof variation expressed as a percent, where n = 3. NC: Not calculated.

TABLE 14 Mean Concentration of Drug-Derived Radioactivity (μg EM-800equiv/g tissue) in Selected Tissues of Female Long-Evans Rats Followinga Single Oral Dose of ¹⁴C-EM-800 (20 mg/kg)^(a) Time Brain Liver UterusBlood Plasma (hr) Mean^(b) (% CV) Mean^(b) (% CV) Mean^(b) (% CV)Mean^(b) (% CV) Mean^(b) (% CV) 2 0.0176 (29) 7.2547 (30) 0.3675 (36)0.1487 (22) 0.2246 (20) 4 0.0171 (29) 3.2201 (48) 0.2866 (83) 0.0669(45) 0.1032 (39) 6 0.0156 (8) 2.7462 (8 0.2757 (19) 0.0483 (29) 0.0741(31) 8 0.0168 (31) 2.7748 (8) 0.3332 (46) 0.0411 (12) 0.0641 (14) 120.0100^(c) (87) 1.8232 (38) 0.2407 (25) 0.0287 (54) 0.0407 (55) 24 0(NC) 0.6391 (52) 0.0837 (54) 0.0047^(d) (173) 0.0101 (86) 36 0 (NC)0.4034 (22) 0.0261 (15) 0 (NC) 0 (NC) 48 0 (NC) 0.2196 (37) 0.0238 (44)0 (NC) 0 (NC) 72 0 (NC) 0.1326 (4) 0 (NC) 0 (NC) 0 (NC) 96 0 (NC) 0.0944(15) 0 (NC) 0 (NC) 0 (NC) 168 0 (NC) 0.0348 (14) 0 (NC) 0 (NC) 0 (NC)^(a)Values from report tables for LREM 1129 (EM-800: Tissue Distributionand Excretion of Radioactivity Following a Single Oral Dose of¹⁴C-EM-800 (20 mg/2 mL/kg) to Male and Female Long-Evans Rats).^(b)Limit of quantification (LOQ) of 1.2 ng EM-800 equivalent. ^(c)Onesample below the LOQ; 0 used in calculation of mean. ^(d)Two samplesbelow the LOQ; 0 used in calculation of mean. % CV: Coefficient ofvariation expressed as a percent, where n = 3. NC: Not calculated.

Example 9 Animals

Female BALB/c mice (BALB/cAnNCrlBR), approximately 50 days old andweighing 18-20 g, were obtained from Charles-River, Inc. (St-Constant,Quebec, Canada) and housed 4-5/cage in a temperature (23±1° C.)- andlight (12 h light/day, lights on at 7:15)-controlled environment. Themice were fed rodent chow and tap water ad libitum. The animals wereovariectomized (OVX) under general anesthesia (Avertin) via bilateralflank incisions and randomly assigned to groups of 9-10 animals.

Treatments

CS-115-1 (racemic EM-652) and EM-762 (racemic EM-800) were administeredorally by gavage or by topical application on the dorsal skin once dailyat different doses, namely 0.75, 2.5, 7.5, 25 or 75 nmol ofcompound/gavage or application/animal. Treatment with the antiestrogens(0.2 mL/mouse/gavage or application) was initiated 2 days afterovariectomy, while treatment with estrone (0.06 μg, subcutaneousinjection (s.c.), twice daily) was started 3 days later (5 dayspost-ovariectomy). Thereafter, estrone and antiestrogens wereadministered in combination for a 6 day-period. For oral administration,compounds were dissolved in a 50:50 (vol/vol) mixture of polyethyleneglycol 600 (PEG-600) and ethanol and administered in a 1% (w/v)gelatin-0.9% NaCl solution (final concentration of PEG-600:ETOH was 8%)while for the percutaneous administration, compounds were solubilized in50% ETOH-50% propylene glycol. Mice in the OVX control group receivedthe oral vehicle alone during the 9-day period. The animals were killedby cervical dislocation on the 11^(th) morning following ovariectomy.Uteri were rapidly dissected and weighed.

As can be seen on FIG. 16, comparable effects are observed afteradministration of acolbifene derivatives by the oral and percutaneousroutes.

Pharmaceutical Composition Examples

Set forth below, by way of example and not of limitation, are severalpharmaceutical compositions utilizing preferred active SERM acolbifene(EM-652.HCl; EM-1538) and preferred active sex steroid precursordehydroepiandrosterone (DHEA, Prasterone). Other compounds of theinvention or combination thereof, may be used in place of (or inaddition to) acolbifene or dehydroepiandrosterone. The concentration ofactive ingredient may be varied over a wide range as discussed herein.The amounts and types of other ingredients that may be included are wellknown in the art.

Example A Pharmaceutical Composition for Orally Administration(Capsules)

Weight % Ingredient (by weight of total composition) Acolbifene 5.0 DHEA10.0 Lactose hydrous 70.0 Starch 4.8 Cellulose microcrystalline 9.8Magnesium stearate 0.4

Example B Pharmaceutical Composition for Orally Administration (Tablets)

Weight % Ingredient (by weight of total composition) Acolbifene 5.0 DHEA15.0 Gelatin 5.0 Lactose 58.5 Starch 16.5

Example C Topical Administration (Cream)

Weight % Ingredient (by weight of total composition) DHEA 1.0 Acolbifene0.2 Emulsifying Wax, NF 18.0 Light mineral oil, NF 12.0 Benzyl alcohol1.0 Ethanol 95% USP 33.8 Purifed water, USP 34.0

Example D Rectal Administration Rectal Suppository or Ovule

Weight % Ingredient (by weight of total composition) DHEA 0.25 to 2.0Acolbifene 0.25 to 3.0 Witepsol H-15 base  95.0 to 99.5

DHEA suppositories were prepared using Witepsol H-15 base (Medisca,Montreal, Canada). Any other lipophilic base such as Hard Fat,Fattibase, Wecobee, cocoa butter, theobroma oil or other combinations ofWitepsol bases could used. Preferred SERMs are EM-800 and acolbifene

Kit Examples

Set forth below, by way of example and not of limitation, are severalkits utilizing preferred active SERM acolbifene, preferred antiestrogenFaslodex and preferred active a sex steroid precursor DHEA. Theconcentration of active ingredient may be varied over a wide range asdiscussed herein. The amounts and types of other ingredients that may beincluded are well known in the art.

Example D Kit The SERM and Sex Steroid Precursor are Orally AdministeredSERM Composition for Oral Administration (Capsules)

Weight % Ingredient (by weight of total composition) Acolbifene 5.0Lactose hydrous 80.0 Starch 4.8 Cellulose microcrystalline 9.8 Magnesiumstearate 0.4

+ DHEA Composition for Oral Administration (Gelatin Capsule)

Weight % Ingredient (by weight of total composition) DHEA 25.0 Lactosehydrous 27.2 Sodium Starch Glycolate 20.0 Microcrystalline Cellulose,Colloidal 27.2 Silicon Dioxide, Silica Colloidal Anhydrous and LightAnhydrous Silicic Acid Colloidal Silicon Dioxide 0.1 Magnesium stearate0.5

Other SERMs may be substituted for acolbifene in the above formulations,as well as other sex steroid precursors may be substituted for DHEA.More than one SERM or more than one sex steroid precursor may beincluded in which case the combined weight percentage is preferably thatof the weight percentage for the single sex steroid precursor or singleSERM given in the examples above.

Example E Kit The SERM is Orally Administered and the Sex SteroidPrecursor is Rectally Administered SERM Composition for OralAdministration (Capsules)

Weight % Ingredient (by weight of total composition) Acolbifene 5.0Lactose hydrous 80.0 Starch 4.8 Cellulose microcrystalline 9.8 Magnesiumstearate 0.4

+ Rectal Suppository

Weight % Ingredient (by weight of total composition) DHEA 0.25 to 2.0   Witepsol H-15 base 98 to 99.75

DHEA suppositories were prepared using Witepsol H-15 base (Medisca,Montreal, Canada). Any other lipophilic base such as Hard Fat,Fattibase, Wecobee, cocoa butter, theobroma oil or other combinations ofWitepsol bases could used.

Example F Kit The SERM and the Sex Steroid Precursor are RectallyAdministered Rectal Suppository

Weight % Ingredient (by weight of total composition) DHEA 0.25 to 2.0   Witepsol H-15 base 98 to 99.75

+ Rectal Suppository

Weight % Ingredient (by weight of total composition) Acolbifene 0.3 to3.0 Hard Fat 97.0 to 99.7

Acolbifene suppositories were prepared using Hard Fat (Witepsol). Anyother bases such as Fattibase, Wecobee, cocoa butter, theobroma oil orother combinations of Hard Fat could be used.

Example G The SERM is Orally Administered and the Sex Steroid Precursoris Percutaneously Administered SERM Composition for Oral Administration(Capsules)

Weight % Ingredient (by weight of total composition) Acolbifene 5.0Lactose hydrous 80.0 Starch 4.8 Cellulose microcrystalline 9.8 Magnesiumstearate 0.4

+ Sex Steroid Precursor Composition for Transdermal Administration (Gel)

Weight % (by weight of total Ingredient composition) DHEA 2.0Caprylic-capric Triglyceride (Neobee M-5) 5.0 Hexylene Glycol 15.0Transcutol (diethyleneglycol monomethyl ether) 5.0 Benzyl alcohol 2.0Cyclomethicone (Dow corning 345) 5.0 Ethanol (absolute) 64.0Hydroxypropylcellulose (1500 cps) (KLUCEL) 2.0

or Sex Steroid Precursor Composition for Transdermal Administration(Cream)

Weight % (by weight of total composition) Ingredient FormulationEM-760-48-1.0% Cyclometicone 5.0% Light mineral oil 3.0% 2-ethylhexylstearate 10.0%  Cutina E24 1.0% DC emulsifier 10 3.0% BHT 0.09% Propyleneglycol 46.01%  Ethanol 95 10.0%  DHEA 1.0% Eau purifiee 15.0% MgSO4 0.65%  Ethanol 95 5.25%  Total 100.0% 

Example H Kit The Antiestrogen is Intramuscularly Administered and SexSteroid Precursor is Orally Administered Commercially AvailableSteroidal Antiestrogen Faslodex + DHEA Composition for OralAdministration (Gelatin Capsule)

Weight % (by weight of total Ingredient composition) DHEA 25.0 Lactosehydrous 27.2 Sodium Starch Glycolate 20.0 Microcrystalline Cellulose,Colloidal Silicon 27.2 Dioxide, Silica Colloidal Anhydrous and LightAnhydrous Silicic Acid Colloidal Silicon Dioxide 0.1 Magnesium stearate0.5

Other SERMs (toremifene, ospemifene, raloxifene, arzoxifene,lasofoxifene, TSE-424, ERA-923, EM-800, SERM 3339, GW-5638) may besubstituted for acolbifene in the above formulations, as well as othersex steroid precursors may be substituted for DHEA. More than one SERMor more than one precursor may be included in which case the combinedweight percentage is preferably that of the weight percentage for thesingle precursor or single SERM given in the examples above.

The invention has been described in terms of preferred embodiments andexamples, but is not limited thereby. Those of skill in the art willreadily recognize the broader applicability and scope of the inventionwhich is limited only by the patent claims herein.

Recommandations

We suggest men having a palpable prostatic nodule or induration or witha serum PSA above 3 ng/mL to have further urological evaluation beforetreatment as suggested by the Guidelines of the Endocrine Society(Bhasin, Cunningham et al. 2006).

Similarly, treatment is not recommended in men with erythrocytosis(hematocrit >50%), untreated obstructive sleep apnea, severe untreatedbenign prostatic hyperplasia with IPSS score >19 or uncontrolled heartfailure.

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1. A method of preventing, reducing or eliminating the incidence of maleandrogen deficiency symptoms or diseases including malehypogonadism-associated symptoms and diseases, said method comprisingadministering to male patient in need of said prevention, reduction orelimination, (i) a therapeutically effective amount of a sex steroidprecursor or prodrug thereof in association with (ii) a therapeuticallyeffective amount of a selective estrogen receptor modulator or anantiestrogen or prodrug of either, wherein the selective estrogenreceptor modulator or antiestrogen stimulates LH secretion whichincreases the level of circulating testosterone.
 2. The method of claim1 wherein the symptoms or diseases are associated with low testosterone,low DHEA or both.
 3. The method of claim 1 wherein the symptoms ordiseases are selected from the group comprising of loss of libido,erectile dysfunction, tiredness, loss of energy, depression, bone loss,muscle loss, muscle weakness, fat accumulation, memory loss, cognitionloss, Alzheimer's disease, dementia, loss of body hair, fertilityproblems, insomnia, gynecomastia, anemia, hot flushes, sweats, decreasedsense of well-being, obesity, osteoporosis, hypercholesterolemia,hyperlipidemia, atherosclerosis, hypertension, insulin resistance,cardiovascular disease and type 2 diabetes.
 4. The method of claim 1wherein the sex steroid precursor is selected from the group consistingof dehydroepiandrosterone, dehydroepiandrosterone-sulfate,androst-5-ene-3β,17β-diol and 4-androstene-3,17-dione.
 5. The method ofclaim 1 wherein the sex steroid precursor is dehydroepiandrosterone. 6.The method of claim 1 wherein the selective estrogen receptor modulatoris selected from the group comprising of Tamoxifen, Toremifene, CC 8490,SERM 3471, HMR 3339, HMR 3656, Raloxifene, LY 335124, LY 326315,Arzoxifene (LY 353381), Pipendoxifene (ERA 923), Bazedoxifene (TSE 424,WAY 140424), Oporia (Lasofoxifene), EM-652, EM-800, EM-652-HCl(acolbifene, EM-1538), 4-hydroxy-Tamoxifen, 4-hydroxy-Toremifene,Droloxifene, LY 335563, GW-5638, Idoxifene, Levormeloxifene, Iproxifen(TAT-59), Ospemifene (FC 1271), Fispemifene, Centchroman, CHF 4227, LY2066948, LY 2120310, Sivifene, SR 16234, Clomiphene, Enclomiphene,Zuclomiphene, GW 7603, BL 3040, SRI 16158, SR 16157, SRI 16137, SR16137, Rad 1901,(+)-3-(4-hydroxyphenyl)-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-4-(trifluoromethyl)-2H-1-benzopyran-7-ol,Femarelle, Nafoxidine and Endoxifen.
 7. The method of claim 1 whereinthe selective estrogen receptor modulator has the following formula:

a) wherein R₁ and R₂ are independently hydrogen, hydroxyl, halogen,C₁-C₆ alkyl or a moiety which is converted to hydroxyl in vivo; b)wherein Z is either absent or selected from the group consisting of—CH₂—, —O—, —S— and —NR₃— (R₃ being hydrogen or C₁-C₆ alkyl); c) whereinthe R₁₀₀ is a bivalent moiety which distances L from the B-ring by 4-10intervening atoms; d) wherein L is a bivalent or trivalent moietyselected from the group of —SO—, —CON<, —N<, and —SON<; e) wherein G₁ isselected from the group consisting of hydrogen, a C₁ to C₅ hydrocarbon,a bivalent moiety which in combination with G₂ and L is a 5- to7-membered heterocyclic ring, and halo or unsaturated derivatives of theforegoing; f) wherein G₂ is either absent or selected from the groupconsisting of hydrogen, a C₁ to C₅ hydrocarbon, a bivalent moiety whichin combination with G₁ and L is a 5- to 7-membered heterocyclic ring,and halo or unsaturated derivatives of the foregoing; g) wherein G₃ isselected from the group consisting of hydrogen, methyl, ethyl andtrifluoromethyl.
 8. The method of claim 1, wherein the selectiveestrogen receptor modulator is a benzopyran compound of the followinggeneral structure:

or a pharmaceutically acceptable salt thereof, a) wherein D is—OCH₂CH₂N(R₃)R₄ (R₃ and R₄ either being independently selected from thegroup consisting of C₁-C₄ alkyl, or R₃, R₄ and the nitrogen atom towhich they are bound, together being a ring structure selected from thegroup consisting of pyrrolidinyl, 2,2-dimethylpyrrolidinyl,2-methylpyrrolidinyl, piperidino, hexamethyleneimino, and morpholino);b) wherein R₁ and R₂ are independently selected from the groupconsisting of: hydrogen, hydroxyl, halogen, C₁-C₆ alkyl, and a moietyconverted in vivo to hydroxyl; c) wherein G₃ is selected from the groupconsisting of hydrogen, methyl, ethyl and trifluoromethyl.
 9. The methodof claim 1, wherein the selective estrogen receptor modulator is abenzopyran compound which is an optically active compound having anabsolute configuration S on carbon 2, said compound having the molecularstructure:

or a pharmaceutically acceptable salt thereof; a) wherein R₁ and R₂ areindependently selected from the group consisting of hydroxyl, halogen,C₁-C₆ alkyl, and a moiety convertible in vivo to hydroxyl; b) wherein R³is a species selected from the group consisting of saturated,unsaturated or substituted pyrrolidinyl, saturated, unsaturated orsubstituted piperidino, saturated, unsaturated or substitutedpiperidinyl, saturated, unsaturated or substituted morpholino,nitrogen-containing cyclic moiety, nitrogen-containing polycyclicmoiety, and NRaRb (Ra and Rb being independently hydrogen, straight orbranched C₁-C₆ alkyl, straight or branched C₂-C₆ alkenyl, or straight orbranched C₂-C₆ alkynyl).
 10. The method of claim 1 where the selectiveestrogen receptor modulator is acolbifene:

and is an optically active compound having an absolute configuration Son carbon
 2. 11. The method of claim 9 wherein the selective estrogenreceptor modulator is a benzopyran salt of an acid selected from thegroup consisting of acetic acid, adipic acid, benzenesulfonic acid,benzoic acid, camphorsulfonic acid, citric acid, fumaric acid,hydroiodic acid, hydrobromic acid, hydrochloric acid,hydrochlorothiazide acid, hydroxy-naphthoic acid, lactic acid, maleicacid, methanesulfonic acid, methylsulfuric acid,1,5-naphthalenedisulfonic acid, nitric acid, palmitic acid, pivalicacid, phosphoric acid, propionic acid, succinic acid, sulfuric acid,tartaric acid, terephthalic acid, p-toluenesulfonic acid, and valericacid.
 12. The method of claim 1 wherein the selective estrogen receptormodulator is acolbifene:

and is an optically active compound having an absolute configuration Son carbon 2; and wherein the sex steroid precursor isdehydroepiandrosterone.
 13. The method of claim 1, wherein theantiestrogen is selected from the group comprising of Faslodex (ICI182780, fulvestrant,7α-[9-(4,4,5,5,5-pentafluoro-pentylsulphinyl)nonyl]oestra-1,3,5(10)-triene-3,17β-diol),ICI 164384, CH 4893237, ZK 246965 and SH
 646. 14. The method of claim 1,wherein the selective estrogen receptor modulator has no estrogenicactivity in breast, uterine or endometrial tissues.
 15. The method ofclaim 1 which reduces the risk of the male patients acquiring breastcancer.
 16. The method of claim 1, further comprising administering aspart of a combination therapy, a therapeutically effective amount ofhuman chorionic gonadotropin.
 17. The method of claim 1 wherein theselective estrogen receptor modulator and/or sex steroid precursorare/is rectally administered.
 18. The method of claim 1 wherein theselective estrogen receptor modulator and/or sex steroid precursorare/is orally administered.
 19. The method of claim 1 wherein theselective estrogen receptor modulator and/or sex steroid precursorare/is percutaneously administered.
 20. A pharmaceutical composition forpreventing, reducing or eliminating the incidence of male androgendeficiency symptoms or diseases including male hypogonadism-associatedsymptoms and diseases as defined in claim 3, comprising: a) apharmaceutically acceptable excipient, diluent or carrier; b) at leastone sex steroid precursor or prodrug thereof as defined in claim 4; andc) at least one selective estrogen receptor modulator or an antiestrogenor prodrug of either as defined in any of claim 6-11 or 13; wherein saidpharmaceutical composition is provided in packaging that directs use ofsaid composition for prevention, reduction or elimination of at leastone male androgen deficiency symptom or disease.
 21. The pharmaceuticalcomposition of claim 20 wherein both sex steroid precursor and selectiveestrogen receptor modulator or antiestrogen are formulated together in apharmaceutical delivery form selected from the group comprising of pill,tablet, capsule, cream, gel, rectal suppository and injection.
 22. Thepharmaceutical composition of claim 20 wherein the selective estrogenreceptor modulator is acolbifene:

and is an optically active compound having an absolute configuration Sat carbon 2; and wherein the sex steroid precursor isdehydroepiandrosterone.
 23. A kit for preventing, reducing oreliminating the incidence of male androgen deficiency symptoms ordiseases including male hypogonadism-associated symptoms and diseases asdefined in claim 3, comprising (i) a first container having therein atleast one sex steroid precursor or a prodrug thereof as defined in claim4; (ii) a second container having therein at least one selectiveestrogen receptor modulator, or an antiestrogen or prodrug of either ofthe foregoing as defined in any of claims 6-11 or 13; and (iii)instructions for using the kit for the prevention, reduction orelimination of at least one male androgen deficiency symptom or disease.24. The kit of claim 23 wherein the selective estrogen receptormodulator is acolbifene:

and is an optically active compound having an absolute configuration Son carbon 2; and wherein the sex steroid precursor isdehydroepiandrosterone.